Modified peptides and associated methods of use

ABSTRACT

The present disclosure relates to a modified peptide including (i) an amino acid sequence (X)-GRT-(Y)-TLC-(Z), or (ii) an amino acid sequence having at least 40% sequence identity to the amino acid sequence (X)-GRT-(Y)-TLC-(Z), wherein X, Y, and Z are the same as described in the specification. In this regard, methods for inhibiting the activity of at least one enzyme selected from the group consisting of AKT1 (PKB alpha), AKT2 (PKB beta), MAP3K8 (COT), MST4, AURKB (Aurora B), ROCK1, RPS6KB1 (p70S6K), CDC42 BPA (MRCKA), BRAF, RAF1 (cRAF) Y340D Y341D, SGK (SGK1), MAP4K4 (HGK), AURKA (Aurora A), AURKC (Aurora C), BRAF V599E, CHEK1 (CHK1), GSG2 (Haspin), CHEK2 (CHK2), FGR, IKBKB (IKK beta), CDK7/cyclin H/MNAT1, and CDC42 BPB (MRCKB) and Abl; and inhibiting cell proliferation are also provided, as are methods for preventing or treating cancer or a neurodegenerative disease or disorder.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present disclosure is claims priority to and the benefit of U.S.Provisional Patent Application No. 62/857,293, filed 5 Jun. 2019 andtilted: MODIFIED PEPTIDES AND ASSOCIATED METHODS OF USE, which isincorporated herein by reference in its entirety for all purposes.

INCORPORATION BY REFERENCE

In compliance with 37 C.F.R. 1.52(e)(5), the sequence informationcontained in electronic file name: ADN0002US2_Sequence_Listing_25SEP2020 Revised.txt; size 59.6 KB (61,112 bytes); created on: 27 Nov.2020, is incorporated herein by reference in its entirety.

BACKGROUND

1. Field of the discovery. The present disclosure generally relates tomodified therapeutic polypeptides, their compositions, and methods ofadministration to an organism in need thereof for treating and/orpreventing disease, for example, cancer.

2. Background information. The AGC serine/threonine protein family ofkinases consists of 63 evolutionarily related kinases, including PDK1,PKB/AKT, SGK, PKC, PRK/PKN, MSK, RSK, S6K, PKA, PKG, DMPK, MRCK, ROCK,NDR, LATS, CRIK, MAST, GRK, Sgk494, YANK, Aurora and PLK. The differentAGC kinase families share several aspects of their mechanisms ofinhibition and activation. The conformation of the catalytic domain ofmany AGC kinases is regulated by the modulation of the conformation of aregulatory site on the small lobe of the kinase domain, the PIF-pocket.The PIF-pocket acts like an ON-OFF switch in AGC kinases with differentmodes of regulation, i.e. PDK1, PKB/AKT, LATS and Aurora kinases.Molecular probes stabilizing the PIF-pocket in the active conformationare activators, while compounds stabilizing the disrupted site areallosteric inhibitors. (Leroux et al. (2018) Semin Cancer Biol 48:1-17.Doi: 10.1016).

The serine-threonine kinase AKT (known also as Protein Kinase B)phosphorylates various protein substrates to regulate many keyphysiological processes, such as cell cycle, glucose metabolism, cellgrowth and survival, angiogenesis and protein synthesis (Brazil, et al.(2002) Cell 111:293-303). Stimulation of its catalytic activity istriggered by phosphatidylinositol 3 kinase and results from thePtdIns(3,4,5)P-dependent recruitment of AKT, from the cytoplasm to themembrane, as well as the phosphorylation of two regulatory residues,Thr-308 and Ser-473. Phosphorylation of Thr-308, catalyzed by PDK-1, isrequired for AKT activity, and this activity is augmented, ˜10 fold, bySer-473 phosphorylation (Alessi, et al. (1996) EMBO J. 15:6541-6551;Brazil, et al. (2002) supra).

Protein Kinase A (PKA) is ubiquitously expressed in mammalian cells andregulates important cellular processes such as growth, development,memory, metabolism, gene expression, and lipolysis. The PKA holoenzymeexists as an inactive complex and is composed of two catalytic (PKAc)and regulatory (PKA RI & RII) subunits. Binding of cAMP facilitates thedissociation and activation of catalytic subunits. Each catalyticsubunit is composed of a small and large lobe, with the active siteforming a cleft between the two lobes. The small lobe provides thebinding site for ATP, and the large lobe provides catalytic residues anda docking surface for peptide/protein substrates. The activation loop inthe large lobe contains a phosphorylation site, Thr-197, which isessential for catalysis (Adams, et al. (1995) Biochemistry34:2447-2454).

Deregulation of AKT signaling pathway is known to be directly associatedwith some of the most prevalent and incurable human disorders such ascancer, neurodegenerative and psychiatric brain disorders, andinfectious diseases (Blain and Massague (2002) Nat. Med. 8:1076-1078;Brazil, et al. (2004) Trends Biochem. Sci. 29:233-242; Chen, et al.(2003) Cell 113:457-468; Colin, et al. (2005) Eur. J. Neurosci.21:1478-1488; Emamian, et al. (2004) Nat. Genetics 36:131-137; Griffin,et al. (2005) J. Neurochem. 93:105-117; Liang, et al. (2002) Nat. Med.8:1153-1160; Shin, et al. (2002) Nat. Med. 8:1145-1152; Viglietto, etal. (2002) Nat. Med. 8:1136-1144; Ji & Liu (2008) Recent Pat Biotechnol.(3):218-26). It is well-established that the hyperactivity of AKT ispart of the pathologic process in several types of the most prevalenthuman malignancies (Brazil, et al. (2004) supra), including breastcancer, prostate cancer, lung cancer, gastrointestinal tumors,pancreatic cancer, hepatocellular carcinoma, thyroid cancer, and centralnervous system malignancies (such as glioblastoma and gliomas).Association of AKT function with several neurodegenerative braindisorders such as the Alzheimer's disease (AD), Huntington's disease(HD), spinocerebellar ataxia type 1 (SCA1), and amyotrophic lateralsclerosis (ALS), have also been reported (Griffin, et al. (2005) supra;Colin, et al. (2005) supra; Saudou, et al. (1998) Cell 95:55-66; Chen,et al. (2003) supra; Emamian, et al. (2003) Neuron 38:375-387; Kaspar,et al. (2003) Science 301:839-842). Moreover, several studies have shownthat activating PI3K-AKT signaling is a strategy used by viruses to slowdown apoptosis and prolong viral replication in both acute andpersistent infection. It is also probable that prevention of cell deathfacilitates virus-induced carcinogenesis. Accumulating evidence suggeststhat the activity of PI3K or AKT is critical for survival of a fewviruses, including HIV and other type of viruses (Ji & Liu (2008) RecentPat Biotechnol. (3):218-26; Chugh, et al. (2008) Retrovirology vol. 511. doi: 10.1186/1742-4690-5-11)

An impairment in the AKT signaling pathway is also involved inschizophrenia (Emamian, et al. (2004) supra). The genetic association ofAKT1 gene with schizophrenia has been identified in European (Schwab, etal. (2005) Biol. Psychiatry 58:446-450) and Japanese (Ikeda, et al.(2004) Biol. Psychiatry 56:698-700) populations. Moreover, the PKAsignaling pathway has been found to mediate the interaction of DISC1 andPDE4B, genetic factors known to be associated with higher risk forschizophrenia (Millar, et al. (2005) Science 310:1187-1191).

Given the association of AKT with some of the most prevalent andincurable human diseases, including cancer, infectious diseases,neurodegenerative and psychiatric disorders, there is a need in the artto identify agents which interact with and modulate the activity of AKT.The present disclosure meets this need in the art.

SUMMARY

Presently described are active therapeutic peptides that can target thecatalytic activity of several mammalian kinases, including severalmembers of the AGC family of kinases, in order to modulate a widevariety of cellular functions, including but not limited to cellproliferation, cell survival, cell death, etc.

In an aspect, the disclosure provides a modified peptide including (i)an amino acid sequence (X)-GRT-(Y)-TLC-(Z), or (ii) an amino acidsequence having at least 40% sequence identity to the amino acidsequence (X)-GRT-(Y)-TLC-(Z). In these formulae, X is a natural aminoacid, a non-natural amino acid, a chemical modification of a natural ornon-natural amino acid, an acetyl group, a lipid group, or a combinationthereof; Y is a natural amino acid, a non-natural amino acid, a chemicalmodification of a natural or non-natural amino acid, or a combinationthereof; and Z is a natural amino acid, a non-natural amino acid, achemical modification of a natural or non-natural amino acid, an aminegroup, or a combination thereof.

The disclosure also provides modified peptides having the formula(X)-(seq1)-(Y)-(seq2)-(Z) or an amino acid sequence having at least 40%,50%, 60%, 70%, 80%, 90% or more sequence identity to the amino acidsequence (X)-(seq1)-(Y)-(seq2)-(Z). In these formulae, seq1 is GRT,KGRT, VKGRT, RVKGRT, KRVKGRT, (Orn)-RVKGRT, or AKRVKGRT; seq2 is TLC,TLCG, TLCGR, TLCGRPE, TLCGRPEY, or TLCGRPE-(4-Cl-Phe); X is a naturalamino acid, a non-natural amino acid, a chemical modification of anatural or non-natural amino acid, an acetyl group, a lipid group, or acombination thereof; Y is a natural amino acid, a non-natural aminoacid, a chemical modification of a natural or non-natural amino acid, ora combination thereof; and Z is a natural amino acid, a non-naturalamino acid, a chemical modification of a natural or non-natural aminoacid, an amine group, or a combination thereof.

Also included are methods for inhibiting the activity of at least oneenzyme selected from the group consisting of AKT1 (PKB alpha), AKT2 (PKBbeta), MAP3K8 (COT), MST4, AURKB (Aurora B), ROCK1, RPS6KB1 (p70S6K),CDC42 BPA (MRCKA), BRAF, RAF1 (cRAF) Y340D Y341D, SGK (SGK1), MAP4K4(HGK), AURKA (Aurora A), AURKC (Aurora C), BRAF V599E, CHEK1 (CHK1),GSG2 (Haspin), CHEK2 (CHK2), FGR, IKBKB (IKK beta), CDK7/cyclin H/MNAT1,and CDC42 BPB (MRCKB), and Abl; and inhibiting cell proliferation arealso provided, as are methods for preventing or treating cancer,infectious diseases, or a neurodegenerative disease or disorder.

The preceding general areas of utility are given by way of example onlyand are not intended to be limiting on the scope of the presentdisclosure and appended claims. Additional objects and advantagesassociated with the compositions, methods, and processes of the presentdisclosure will be appreciated by one of ordinary skill in the art inlight of the instant claims, description, and examples. For example, thevarious aspects and embodiments of the present disclosure may beutilized in numerous combinations, all of which are expresslycontemplated by the present description. These additional advantagesobjects and embodiments are expressly included within the scope of thepresent disclosure. The publications and other materials used herein toilluminate the background of the present disclosure, and in particularcases, to provide additional details respecting the practice, areincorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and form a partof the specification, illustrate several embodiments of the presentdisclosure and, together with the description, serve to explain theprinciples of the present disclosure. The drawings are only for thepurpose of illustrating an embodiment of the present disclosure and arenot to be construed as limiting the present disclosure. Further objects,features and advantages of the inventions of the present disclosure willbecome apparent from the following detailed description taken inconjunction with the accompanying figures showing illustrativeembodiments of the present disclosure, in which:

FIG. 1A and FIG. 1B. Intracellular molecular targets of the Exemplarycompounds: (1A) Representative immunoblots from a cell-based assays byprobing with antibodies that recognize a PI3K-P110, or phospho-PDK1(Ser-241), total AKT1, or phospho-P53 (Ser-46). Cells based assays aftertreating the U251 human glioblastoma cells with different concentrationsof vehicle, or an Exemplary compound from 5 uM to 40 uM at differenttime intervals of 30 min, 2 hours or 24 hours. (1B) Representativeimmunoblots of cell based assays similar to A probed with a differentset of primary antibodies including phospho-AKT1 (Thr-308), p-CRAF(Ser-259), phospho-Aurora A (Thr-288), or total Aurora A.

FIG. 2. Apoptosis and survival of U251 glioblastoma cells at differenttime intervals following the treatment with an Exemplary compound: Equalnumber of U251 human glioblastoma cells (5×10⁵) were plated oncoverslips and treated with either vehicle (top panel) or with anExemplary compound (bottom panels) for 20 minutes, one, two or threedays. Fluorescent TUNEL (green) assay was performed to visualize theapoptotic cells and DAPI staining was used to visualize the nuclei ofall cells (blue). Indirect immunofluorescent staining of Cleaved-Caspase3 (red) was performed as another marker of apoptosis. Cells treated withthe vehicle grew to a much higher confluency compared to those treatedwith the Exemplary compound (compared the DAPI signal of the top andbottom panels). The confocal image (20×) is the same Z step showingthree channels separately (the first three columns) and the merged image(last column). The images show a time dependent increase in apoptosisrate of the cells after treatment, after 3 days of treatment almost 100%of cells Exemplary compound are apoptotic shown by both Tunel (green)and increased Cleaved-Caspase 3 (red).

FIG. 3A and FIG. 3B. Quantitative analysis of the cell density andapoptosis rate at different time intervals: Equal cell numbers wereplated on coverslips and were treated with an Exemplary compound andvehicle for different time intervals. Cells were stained with DAPI andTunel and analyzed with confocal microscope. FIG. 3A histogram barsreflect the average number of total cells left on coverslips (DAPIpositive cells) counted on five separate confocal fields. FIG. 3Bhistogram bars reflect the average percent of apoptotic cells calculatedby dividing the number of Tunel positive cells to the total cells lefton coverslips (DAPI positive cells) counted on five separate confocalfields. Cells treated with the vehicle constantly showed a higherdensity over time that reached to approximately full confluency at 72hours (not shown), while cells treated with the Exemplary compoundconsistently showed a significant decrease in the cell density over time(3A) and a time-dependent increase in the number of apoptotic cells (3B)to the extent that almost 100% of the cells were apoptotic after 72hours of treatment.

FIG. 4: The IC-50 of the Exemplary compounds in human breast, lung,blood and skin cancer cells: A representative plate of metastatic humanbreast cancer cells (BT-549 and MDA-MB-468), human acute promyelocyticleukemia (HL-60), a Multiple Myeloma cancer cell line (RPMI-8226), asmall-cell lung cancer cell line (DMS-114), a human melanoma cancer cellline (SK-ML-5) after three days treatment with three Exemplarycompounds, the vehicle (PBS), or controls of other kinase inhibitorcompounds in clinical trial (MK-2206), or in clinical use (Imatinib orGleevec), serially diluted from 50 μM to 0.7 μM. This experiment showsthe superior efficacy and a nano-molar range of IC-50 of the exemplarycompounds compared to the existing class of drugs in the market or inlatest stages of the clinical trials.

FIG. 5A, FIG. 5B, and FIG. 5C: Inhibition of tumor growth followed bylocal administration of an Exemplary compound in (5A) an animal modelsof brain tumors (GBM), (5B) an animal model of liver cancer (HCC), andan animal model of metastatic breast cancer: Percentage change in tumorsizes at different time intervals after intratumoral injections (on days0, 3, 7, 11, 14, 17, and 20) of vehicle (n=8) or the Exemplary compound(n=8) treated animals, p values reflect the statistical differencebetween the average tumor size of two groups at the same time intervals,obtained from the Student-T Test or the Mann-Whitney Test.

DETAILED DESCRIPTION

Reference will now be made in detail to exemplary embodiments, examplesof which are illustrated in the present description. In this regard, thepresent exemplary embodiments may have different forms and should not beconstrued as being limited to the descriptions set forth herein.Accordingly, the exemplary embodiments are merely described below, byreferring to the figures, to explain aspects of the present description.As used herein, the term “and/or” includes any and all combinations ofone or more of the associated listed items. Expressions such as “atleast one of,” when preceding a list of elements, modify the entire listof elements and do not modify the individual elements of the list.

It will be understood that, although the terms first, second, third,etc., may be used herein to describe various elements, components,regions, layers, and/or sections, these elements, components, regions,layers, and/or sections should not be limited by these terms. Theseterms are only used to distinguish one element, component, region,layer, or section from another element, component, region, layer, orsection. Thus, a first element, component, region, layer, or sectiondiscussed below could be termed a second element, component, region,layer, or section without departing from the teachings of the presentembodiments.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. As used herein, thesingular forms “a,” “an,” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.

It will be further understood that the terms “comprises” and/or“comprising,” or “includes” and/or “including” when used in thisspecification, specify the presence of stated features, regions,integers, steps, operations, elements, and/or components, but do notpreclude the presence or addition of one or more other features,regions, integers, steps, operations, elements, components, and/orgroups thereof.

“About” as used herein is inclusive of the stated value and means withinan acceptable range of deviation for the particular value as determinedby one of ordinary skill in the art, considering the measurement inquestion and the error associated with measurement of the particularquantity (i.e., the limitations of the measurement system). For example,“about” can mean within one or more standard deviations, or within ±30%,20%, 10%, 5% of the stated value.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which the present disclosure belongs. Itwill be further understood that terms, such as those defined in commonlyused dictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the relevant art and thepresent disclosure, and will not be interpreted in an idealized oroverly formal sense unless expressly so defined herein.

AKT has emerged as the focal point of many signal transduction pathways,regulating multiple cellular processes such as glucose metabolism,transcription, apoptosis, cell proliferation, angiogenesis, and cellmotility (Brazil, et al. (2002) supra). Besides functioning as a kinaseof many substrates involved in these processes, it forms complexes withother proteins that are not substrates, wherein the other proteinsmodulate AKT activity and function (Brazil, et al. (2002) supra).

As used here, the terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably, and refer to any molecule having at least two aminoacids, amino acid analogs or derivatives linked by a peptide bond orother covalent bond.

A physical interaction between AKT and PKAc has been identified.Full-length PKAc was found to potently inhibit the catalytic activity ofAKT, while active AKT increased the catalytic activity of PKA through amechanism that increased the phosphorylation level of PKAc at Thr-197.Unexpectedly, short PKAc fragments could also modulate AKT. Somepeptides were found to activate AKT, while others inhibited AKTactivity. In particular, a PKAc fragment flanking Thr-197 of PKAc,designated herein as ZaTa, was sufficient to potently inhibit AKT invitro and in vivo. ZaTa penetrated into the cell, co-localized with AKT,inhibited and redistributed AKT within the cell, and changed theexpression pattern of PKAc. ZaTa also disrupted the AKT-PKAc complex,both in vitro and in vivo, which resulted in substantial changes inneurite and axon morphology. Treatment of cultured cells with ZaTacaused a dose-dependent inhibition of cell proliferation as well.Furthermore, reducing PKAc protein level increased the AKT protein levelin vitro and in vivo. Accordingly, PKAc and fragments thereof were founduseful for modulating AKT signal transduction pathways involved inregulating glucose metabolism, transcription, apoptosis, cellproliferation, angiogenesis, and cell motility thereby facilitating theprevention or treatment of cancer, infectious diseases, autoimmune,neurodegenerative and psychiatric disorders.

To identify proteins that directly interact with AKT,co-immunoprecipitation assays were performed to purify AKT from thebrain lysate. Co-immunoprecipitated proteins were separated by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) andanalyzed by mass spectrometry. Using this approach, the catalyticsubunit of PKA (PKAc) was identified as an AKT interacting protein. Inseveral independent co-immunoprecipitation experiments using twodifferent antibodies that recognized distinct epitopes on the AKTsequence, i.e., one antibody recognized phosphorylated AKT at Ser-473and the other antibody was raised against the pleckstrin homology (PH)domain of AKT, PKAc was detected in complex with AKT as determined bywestern blot analysis with an antibody against PKAc. Unexpectedly, ahigher amount of PKAc was immunoprecipitated in complex with AKT whenthe sample was treated with cAMP, which increased the level of unboundPKAc to the regulatory subunits. These data demonstrate a physicalinteraction between endogenous PKAc and AKT, wherein the interactionoccurs after activation of PKA.

To demonstrate that PKAc and AKT co-localize, subcellular localizationof PKAc and AKT was analyzed by immunofluorescence confocal microscopy.Neuroblastoma 2a (N2a) neurons expressing an endogenous level of AKT andPKAc were double-labeled with anti-AKT (PH domain) and anti-PKAcantibodies. The strongest signal for the endogenous level of bothmolecules in cultured N2a cells was detected along the neurite and onthe neurite outgrowth zone. However, NG-108 neurons, a somatic cellhybrid of glioblastoma and neuroblastoma, showed a more diffuse patternof co-localization in the cytoplasm and a weaker signal in neurites.These data confirmed the results of the co-immunoprecipitationexperiments and indicated a role for the AKT and PKAc interaction in thegrowth and branching of neuronal cell processes. The co-localization ofAKT and PKAc in non-neuronal cell lines derived from the normal andmalignant cells of human breast tissue was also determined. The celllines analyzed were HTB-126 cells derived from an infiltrating ductalcarcinoma, HTB-125 cells derived from normal breast tissue peripheral tothe infiltrating ductal carcinoma, and CRL-2865 cells derived from thepleural effusion metastatic site of a patient with breast ductalcarcinoma. In these cells, endogenous AKT co-localized with theendogenous PKAc in specific subcellular compartments of both normal andmalignant human breast cells. AKT appeared to co-localize with PKAc in amicrotubule-like structure adjacent to nuclei (both HTB-125 and HTB-126)and on the cell membrane (HTB-125). These data indicated that theAKT-PKAc interaction was not specific to neuronal cells and occurred innormal and malignant cell lines derived from human tissue as well.

To evaluate the significance of the AKT-PKAc interaction, kinaseinhibitors were employed. Highly selective and well-characterized PKAinhibitors and activators are well-known in the art. Thus, culturedneurons were treated with the selective PKA inhibitor, H-89, and thepotent PKA activator, forskolin, and AKT activity was analyzed.Treatment with the PKA inhibitor caused a dose-dependent increase in theactivity of AKT, whereas the PKA activator had an opposing effect,measured by the activation-dependent phosphorylation level of AKT atboth Thr-308 and Ser-473 sites. These findings indicated that the levelof AKT activity in cultured neurons was tightly and inversely correlatedwith the level of PKA activity.

Since the observed effect of H-89 and forskolin on AKT activity incultured cells could be interpreted as the result of the regulatoryinterference of other signaling pathways, in vitro analysis was carriedusing purified, active forms of AKT and PKAc to examine the directresult of this interaction on the kinase catalytic activity. When afull-length, active PKAc was added to AKT kinase assays containingactive AKT as the kinase and Ser-9 GSK-3 glutathione (GST) fusionprotein as the substrate, the kinetic activity of AKT was dramaticallyreduced. This decrease in the catalytic activity of AKT by PKAc wasinhibited when the PKA inhibitor was added to the reaction mixture. Thisobservation indicated that the activity of PKA was required for itsinhibitory effect on AKT. The decrease in the catalytic activity of AKTby PKAc was also observed with two mutants and active forms of AKT1, onewith a deletion in the PH domain and the other one with a Ser473Aspmutation. As with wild-type AKT, the inhibitory effect of PKAc on themutants was reversed in the presence of the PKA inhibitor peptide. Thisindicated that the PH domain of AKT, as well as the phosphorylation atSer-473, were not required for the inhibitory effect of PKAc toward AKT.Further, the same inhibitory effect was observed with active PKAcpurified from bovine tissue, or with human PKAc expressed in Sf9 cells,regardless of the presence or absence of phosphatase inhibitors in thekinase reaction.

The effect of AKT on the catalytic activity of PKA was further analyzedusing an in vitro kinase assay containing PKAc as the kinase andDARPP-32 as the substrate. PKA phosphorylates DARPP-32 at the Thr-34site, converting it into a potent inhibitor of protein phosphatase-1(Huang, et al. (1999) J. Biol. Chem. 274:7870-7878). In contrast to theinhibitory role of PKAc on the AKT catalytic activity, addition of theactive AKT to the PKA kinase assay increased the catalytic activity ofPKAc. This was determined by measuring the phosphorylation level ofDARPP-32 at Thr-34, using a phospho-specific antibody against this site.Unexpectedly, the increase in the PKAc catalytic activity wasaccompanied by an increase in the phosphorylation level of PKAc atThr-197, a residue located in the activation loop of PKAc which isessential for proper biological function and possibly cell motility(Abel, et al. (2001) supra; Cheng, et al. (1998) Proc. Natl. Acad. Sci.USA 95:9849-9854). While autophosphorylation and phosphorylation byPDK-1 have been described as possible mechanisms for thisphosphorylation of Thr-197 in PKAc (Moore, et al. (2002) J. Biol. Chem.277:47878-47884), the in vitro data disclosed herein indicates that AKTphosphorylates Thr-197 of PKAc. Similar opposing effects on thecatalytic activity were observed when kinase reactions were conducted inthe presence of the both PKAc and AKT specific substrates in the samereaction tube. In control assays, neither the phosphorylation of Ser-9GSK-3 GST fusion protein by PKAc, nor the phosphorylation at Thr-34 ofthe recombinant DARPP-32 by AKT was observed.

Unlike PKAc and protein kinase C (PKC) for which potent inhibitorpeptides are readily available and widely used, inhibitors of AKT weregenerally lacking until recent years (Brazil, et al. (2004) supra). Ithas been empirically shown that the use of PKA mutants can facilitatethe structural design of more selective inhibitors for AKT(Breitenlechner, et al. (2005) J. Med. Chem. 48:163-170). Moreover,optimal substrate motifs for AKT have been modified to design AKTinhibitors (Obata, et al. (2000) J. Biol. Chem. 275:36108-36115).Because the full-length PKAc protein inhibited the catalytic activity ofAKT, a peptide library based on the human (GENBANK Accession No.NP_002721; SEQ ID NO:1) and bovine (GENBANK Accession No. CAA47627; SEQID NO:2) PKAc protein sequences was designed and synthesized. Thislibrary contained 96 overlapping peptides (Table 1), covering thefull-length protein sequence of human and bovine PKAc from the N- toC-terminus. The library was extensively screened to identify fragmentsof PKAc that mediated the inhibitory effect of PKAc toward AKT.

TABLE 1 Exemplary Compounds Molec- SEQ Pep- ular ID tide Weight SequenceNO: 1 1577.9 M-G-N-A-A-A-A-K-K-G-S-E-Q-E-S-V 8 2 1651A-A-K-K-G-S-E-Q-E-S-V-K-E-F-L 9 3 1651 G-S-E-Q-E-S-V-K-E-F-L-A-K-A-K 104 1754.2 E-S-V-K-E-F-L-A-K-A-K-E-D-F-L 11 5 1753.3E-F-L-A-K-A-K-E-D-F-L-K-K-W 12 6 1775.2 A-K-A-K-E-D-F-L-K-K-W-E-N-P-A 137 1791 E-D-F-L-K-K-W-E-N-P-A-Q-N-T-A 14 8 1536.7K-K-W-E-N-P-A-Q-N-T-A-H-L 15 9 1670.7 W-E-N-P-A-Q-N-T-A-H-L-D-Q-F 16 101768 P-A-Q-N-T-A-H-L-D-Q-F-E-R-I-K 17 11 1571.9T-A-H-L-D-Q-F-E-R-I-K-T-L 18 12 1849.3 H-L-D-Q-F-E-R-I-K-T-L-G-T-G-S-F19 13 1652.2 E-R-I-K-T-L-G-T-G-S-F-G-R-V-M 20 14 1603.1T-L-G-T-G-S-F-G-R-V-M-L-V-K-H 21 15 1361.8 G-S-F-G-R-V-M-L-V-K-H-M 22 161843.2 S-F-G-R-V-M-L-V-K-H-M-E-T-G-N-H 23 17 1790.2M-L-V-K-H-M-E-T-G-N-H-Y-A-M-K 24 18 1788.2 H-M-E-T-G-N-H-Y-A-M-K-I-L-D-K25 19 1744.2 G-N-H-Y-A-M-K-I-L-D-K-Q-K-V-V 26 20 1642.3A-M-K-I-L-D-K-Q-K-V-V-K-L-K 27 21 1819.3 I-L-D-K-Q-K-V-V-K-L-K-Q-I-E-H28 22 1692.1 K-Q-K-V-V-K-L-K-Q-I-E-H-T-L 29 23 1835.2V-V-K-L-K-Q-I-E-H-T-L-N-E-K-R 30 24 1821.2 K-Q-I-E-H-T-L-N-E-K-R-I-L-Q-A31 25 1683 H-T-L-N-E-K-R-I-L-Q-A-V-N-F 32 26 1788.2N-E-K-R-I-L-Q-A-V-N-F-P-F-L-V 33 27 1778.3 I-L-Q-A-V-N-F-P-F-L-V-K-L-E-F34 28 1715.2 V-N-F-P-F-L-V-K-L-E-F-S-F-K 35 29 1898.4P-F-L-V-K-L-E-F-S-F-K-D-N-S-N-L 36 30 1838.3L-E-F-S-F-K-D-N-S-N-L-Y-M-V-M 37 31 1753.1 F-K-D-N-S-N-L-Y-M-V-M-E-Y-V38 32 1834.2 N-S-N-L-Y-M-V-M-E-Y-V-P-G-G-E-M 39 33 1727.1M-V-M-E-Y-V-P-G-G-E-M-F-S-H-L 40 34 1662.1 Y-V-P-G-G-E-M-F-S-H-L-R-R-I41 35 1663.2 G-G-E-M-F-S-H-L-R-R-I-G-R-F 42 36 1870.3M-F-S-H-L-R-R-I-G-R-F-S-E-P-H 43 37 1905.4 L-R-R-I-G-R-F-S-E-P-H-A-R-F-Y44 38 1750.1 G-R-F-S-E-P-H-A-R-F-Y-A-A-Q-I 45 39 1763.1E-P-H-A-R-F-Y-A-A-Q-I-V-L-T-F 46 40 1871.3 R-F-Y-A-A-Q-I-V-L-T-F-E-Y-L-H47 41 1762.2 A-Q-I-V-L-T-F-E-Y-L-H-S-L-D-L 48 42 1783.3L-T-F-E-Y-L-H-S-L-D-L-I-Y-R 49 43 1778.3 E-Y-L-H-S-L-D-L-I-Y-R-D-L-K 5044 1826.3 H-S-L-D-L-I-Y-R-D-L-K-P-E-N-L 51 45 1600.2L-I-Y-R-D-L-K-P-E-N-L-L-I 52 46 1965.4 Y-R-D-L-K-P-E-N-L-L-I-D-Q-Q-G-Y53 47 1629.9 P-E-N-L-L-I-D-Q-Q-G-Y-I-Q-V 54 48 1653L-L-I-D-Q-Q-G-Y-I-Q-V-T-D-F 55 49 1717 D-Q-Q-G-Y-I-Q-V-T-D-F-G-F-A-K 5650 1672.1 Y-I-Q-V-T-D-F-G-F-A-K-R-V-K 57 51 1768.2V-T-D-F-G-F-A-K-R-V-K-G-R-T-W 58 52 1520 G-F-A-K-R-V-K-G-R-T-W-T-L 59 531966.4 A-K-R-V-K-G-R-T-W-T-L-C-G-T-P-E-Y 60 54 1510.8R-T-W-T-L-C-G-T-P-E-Y-L-A 61 55 1706.1 W-T-L-C-G-T-P-E-Y-L-A-P-E-I-I 6256 1531 G-T-P-E-Y-L-A-P-E-I-I-L-S-K 63 57 1738.3E-Y-L-A-P-E-I-I-L-S-K-G-Y-N-K 64 58 1733.2 P-E-I-I-L-S-K-G-Y-N-K-A-V-D-W65 59 1651.1 L-S-K-G-Y-N-K-A-V-D-W-W-A-L 66 60 1705.2G-Y-N-K-A-V-D-W-W-A-L-G-V-L-I 67 61 1737.2 A-V-D-W-W-A-L-G-V-L-I-Y-E-M-A68 62 1557.1 W-A-L-G-V-L-I-Y-E-M-A-A-G-Y 69 63 1675.2G-V-L-I-Y-E-M-A-A-G-Y-P-P-F-F 70 64 1363.7 Y-E-M-A-A-G-Y-P-P-F-F-A 71 651654 E-M-A-A-G-Y-P-P-F-F-A-D-Q-P-I 72 66 1656G-Y-P-P-F-F-A-D-Q-P-I-Q-I-Y 73 67 1808.2 P-F-F-A-D-Q-P-I-Q-I-Y-E-K-I-V74 68 1717.1 D-Q-P-I-Q-I-Y-E-K-I-V-S-G-K-V 75 69 1680.2I-Q-I-Y-E-K-I-V-S-G-K-V-R-F 76 70 1794.2 Y-E-K-I-V-S-G-K-V-R-F-P-S-H-F77 71 1663 V-S-G-K-V-R-F-P-S-H-F-S-S-D-L 78 72 1761.2V-R-F-P-S-H-F-S-S-D-L-K-D-L-L 79 73 1758.3 S-H-F-S-S-D-L-K-D-L-L-R-N-L-L80 74 1755.3 S-D-L-K-D-L-L-R-N-L-L-Q-V-D-L 81 75 1844.4D-L-L-R-N-L-L-Q-V-D-L-T-K-R-F 82 76 1759.3 N-L-L-Q-V-D-L-T-K-R-F-G-N-L-K83 77 1561 V-D-L-T-K-R-F-G-N-L-K-N-G-V 84 78 1704.2T-K-R-F-G-N-L-K-N-G-V-N-D-I-K 85 79 1737.1 G-N-L-K-N-G-V-N-D-I-K-N-H-K-W86 80 1542.8 N-G-V-N-D-I-K-N-H-K-W-F-A 87 81 1875.1V-N-D-I-K-N-H-K-W-F-A-T-T-D-W 88 82 1894.3 K-N-H-K-W-F-A-T-T-D-W-I-A-I-Y89 83 1898.3 W-F-A-T-T-D-W-I-A-I-Y-Q-R-K-V 90 84 1837.2T-D-W-I-A-I-Y-Q-R-K-V-E-A-P-F 91 85 1807.3 A-I-Y-Q-R-K-V-E-A-P-F-I-P-K-F92 86 1459.9 R-K-V-E-A-P-F-I-P-K-F-K 93 87 1630.1K-V-E-A-P-F-I-P-K-F-K-G-P-G-D 94 88 1652.1 P-F-I-P-K-F-K-G-P-G-D-T-S-N-F95 89 1590.9 K-F-K-G-P-G-D-T-S-N-F-D-D-Y 96 90 1816.9G-P-G-D-T-S-N-F-D-D-Y-E-E-E-E-I 97 91 1845 S-N-F-D-D-Y-E-E-E-E-I-R-V-S-I98 92 1752.9 D-Y-E-E-E-E-I-R-V-S-I-N-E-K 99 93 1633.9E-E-E-I-R-V-S-I-N-E-K-C-G-K 100 94 1886.3I-R-V-S-I-N-E-K-C-G-K-E-F-S-E-F 101 95 1764E-D-F-L-K-K-W-E-S-P-A-Q-N-T-A 102 96 1509.7 K-K-W-E-S-P-A-Q-N-T-A-H-L103 One letter codes used herein include: A, Alanine; R, Arginine; N,Asparagine; D, Aspartate; C, Cysteine; E, Glutamate; Q, Glutamine; G,Glycine; H, Histidine; I, Isoleucine; L, Leucine; K, Lysine; M,Methionine; F, Phenylalanine; P, Proline; S, Serine; T, Threonine; W,Tryptophan; Y, Tyrosine; and V, Valine.

Unexpectedly, individual peptides in the library exhibited significantinhibitory effects toward AKT. These peptides included peptide 49 (SEQID NO:56), 53 (SEQ ID NO:60), 62 (SEQ ID NO:69), 63 (SEQ ID NO:70) and64 (SEQ ID NO:71). Combinations of consecutive overlapping peptidefragments were also assayed for an effect on the catalytic activity ofAKT. A significant inhibitory effect was also observed when peptides 25through 36 were combined (i.e., SEQ ID NOs:32-43), peptides 37 through48 were combined (i.e., SEQ ID NOs:44-55), peptides 49 through 60 werecombined (i.e., SEQ ID NOs:56-67), and peptides 61 through 72 werecombined (i.e., SEQ ID NOs:68-79).

Of particular interest with regard to inhibitory activity toward AKT waspeptideAla-Lys-Arg-Val-Lys-Gly-Arg-Thr-Trp-Thr-Leu-Cys-Gly-Thr-Pro-Glu-Tyr (SEQID NO:60) which flanked the Thr-197 phosphorylation site of PKAc. Thispeptide, designated ZaTa, was sufficient to potently inhibit the invitro catalytic activity of AKT. The phosphorylation level of Ser-9GSK-3 GST substrate was significantly reduced after adding the ZaTapeptide to the AKT1 kinase assay as determined by separating the invitro kinase assay products by SDS-PAGE and western blot analysis with aphospho-specific antibody which specifically recognizes phosphorylatedGSK-3β at Ser-9. Furthermore, the inhibitory effect of ZaTa peptide wascompared with an adjacent peptide(Gly-Phe-Ala-Lys-Arg-Val-Lys-Gly-Arg-Thr-Trp-Thr-Leu; SEQ ID NO:59), apeptide that overlaps with the 11 N-terminal amino acid residues of theZaTa peptide and carries the Thr-197 phosphorylation site. In thisassay, the level of incorporation of γ-³²P into Ser-21 GSK-3β substratepeptide was used as the measure of AKT1 in vitro catalytic activity.While the adjacent overlapping peptide was not able to inhibit AKT1,ZaTa peptide potently inhibited AKT1 catalytic activity in vitro(IC₅₀˜0.1 μM; FIG. 1). ZaTa peptide itself was not a substrate for AKT,as determined by control kinase reactions that contained this peptideand AKT only. This indicated that phosphorylation at Thr-197 by AKTitself was not required for the inhibition of AKT by PKAc and the aminoacid sequence, biochemical characteristics and/or structure flanking theThr-197 site plays a role in inhibiting AKT. ZaTa peptide, which isderived from the native inhibitor of AKT, i.e., PKAc, potently inhibitedAKT and in an independent series of kinase assays did not exhibit anyinhibitory effect on the catalytic activity of PKAc, which, like AKT, isa member of the AGC family of kinases.

Similar to its in vitro inhibitory activity, the ZaTa peptide fragmentwas also able to potently and efficiently inhibit AKT in the brain.After the stereotactic injection of ZaTa peptide, a decrease in thephosphorylation level of AKT substrates in the striatum of a brainhemisphere was observed as compared to the other hemisphere that wasinjected with DMSO as the vehicle. These in vivo immunofluorescenceresults were also confirmed by western blot analyses, which showed asignificant decrease in the phosphorylation level of AKT substrates invivo one hour after the stereotactic injection of ZaTa peptide. As aspecific substrate, the phosphorylation level of GSK-3β at Ser-9 wasalso evaluated. A specific reduction of the phosphorylation level ofGSK-3β at Ser-9 was observed after the stereotactic injection of theZaTa peptide into the striatum of one hemisphere, compared to the otherhemisphere injected with DMSO as the vehicle. The decrease was specificto the AKT phosphorylation site on GSK-3β at Ser-9, since a change inthe phosphorylation level of GSK-3β at Tyr-216 or GSK-3a at Tyr-279 wasnot observed. Moreover, the in vivo reduction of the phosphorylation ofAKT substrates was more obvious at the injection site, since asignificant change in the phosphorylation of AKT substrates in thefrontal cortex or cerebellum between the two hemispheres was notobserved. These data not only confirmed the inhibitory effect of ZaTapeptide on the AKT catalytic activity in vivo, but also demonstrated theefficient distribution and absorption of the ZaTa peptide throughoutbrain tissue.

To test the specificity/selectivity of ZaTa as the inhibitor of AKT1versus the other major kinases, a series of in vitro kinase assays wasperformed at IC₅₀ for AKT1 and at ten times higher concentrations. Apanel of the following 32 kinases was first tested in vitro using theactive form of each kinase and a specific substrate: AKT2, AKT3, PKA,PKCα, PKCγ, PI3Kβ, PI3Kδ, PI3Kγ, SGK, PAK2, PAK3, SAPK2/p38, Abl,CaMKII, CDK1/cyclinB, CDK5/p35, CK1, CK2, CSK, GSK3α, GSK3β, JNK1α1,MAPK1, p70S6K, PDGFRα, PDGFRβ, PDK1, PKG1α, TrkB, JAK2, JAK3, and Syk.At IC₅₀ for AKT1 (0.1 μM), ZaTa showed no significant inhibition on anyof the above kinases in vitro. However, at ten times higherconcentration, ZaTa inhibited AKT2 (63%), PI3Kδ (72%), p70S6K (64%), SGK(73%), PAK3 (83%), JAK3 (79%), TrkB (84%), and Abl (42%). To confirm thein vitro inhibitory effect on these kinases in cells, a cell-based assaywas used to determine the effect of labeled ZaTa on the phosphorylationlevels of well-known intracellular substrates for each kinase (Zipfel,et al. (2004) Curr. Biol. 14:1222-1231; Wang, et al. (2003) Arch.Biochem. Biophys. 410:7-15; King, et al. (1998) Nature 396:180-183;Rangone, et al. (2004) Eur. J. Neurosci. 19:273-279; Middlemas, et al.(1994) J. Biol. Chem. 269:5458-5466; Huang, et al. (1999) J. Biol. Chem.274:7870-7878). The results of this cell-based kinase assay confirmedthat ZaTa could inhibit p70S6K besides AKT. In contrast, intracellularentry of labeled ZaTa did not cause inhibition of PI3K, SGK, PAK3, JAK3,TrkB or Abl, the kinases that ZaTa could inhibit at higherconcentrations in vitro. These data showed that ZaTa had a selectiveinhibitory effect on AKT1 at nanomolar concentrations. However, atmicromolar concentrations ZaTa can also inhibit other select kinases, inparticular p70s6K in cell-based assays. Given that most of theabove-listed kinases have no known inhibitor, it is contemplated thatthe ZaTa could be used at micromolar concentrations in in vitro studiesto inhibit the activity of the select kinases.

To analyze in vivo selectivity, ZaTa was injected into one hemisphereand DMSO in the other hemisphere, as above, and a series of western blotanalyses was performed with phospho-specific antibodies which recognizea phosphorylated substrate or each one of the kinases that ZaTainhibited at higher concentrations in vitro. The results of thisanalysis confirmed potent in vivo inhibition of p70S6K by ZaTa, and aweaker in vivo inhibition of Abl. No in vivo inhibitory effects wereobserved for the other kinases assayed. Given the high functional andstructural homology between p70S6K and AKT, the in vivo inhibitoryeffect of ZaTa on p70S6K was contemplated. Furthermore, the in vivoeffect of ZaTa on the phosphorylation levels of substrates for PKA, PKC,CDKs using phospho-specific antibodies recognizing the phosphorylatedconsensus sites of these kinases was analyzed. In contrast to theconsistent decrease in phosphorylation of AKT substrates, significantchanges in the phosphorylation level of PKA, PKC and CDKs substrates wasnot observed after in vivo injection of ZaTa.

As a striatal specific substrate for PKA and CDK5, the phosphorylationlevel of DARPP-32 was determined at Thr-34 (the PKA site) or at Thr-75(the CDK5 site) (Huang, et al. (1999) supra). ZaTa did not cause anysignificant change in the phosphorylation of DARPP-32 at either of thesesites. Therefore, compared to the other major family of kinasesexpressed in the brain, (i.e., PKA, PKC and CDKs), the ZaTa peptidefragment selectively inhibited AKT in vivo.

Peptides can be very effective inhibitors since they efficiently bind toand inhibit enzymatic activity. However, intracellular delivery ofpeptides can limit their use. With the exception of a few peptides knownas cell-penetrating peptides (CPPs), which have been recognized fortheir use in site-specific drug delivery, inhibitory peptides can havelimited intracellular accumulation in in vivo enzymatic studies. CPPneuropeptides function as neurotransmitters in central and peripheralnervous systems. Based on the primary structure of the ZaTa peptide(i.e., a peptide having a basic arm of several basic residues at theN-terminus and a polar arm composed of several residues with freehydroxyl group at the C-terminus) and in vivo inhibitory effect in thebrain, it was determined whether ZaTa peptide was a CPP. The ZaTapeptide was labeled with a red fluorescent dye at its N-terminus giventhat its C-terminus was important for inhibitory activity. Theefficiency of the labeling and the purity of the labeled peptide wereassessed by mass spectrometry.

The ZaTa peptide was found to penetrate into cells and co-localize withAKT thereby demonstrating that AKT is an intracellular target for ZaTapeptide. The cellular pattern of localization of ZaTa peptide variedfrom cell to cell; some cells showed strong nuclear signals, some showeda cytoplasmic pattern of staining with aggregates, and some showed abright signal on the cell membrane. These different localizationpatterns of ZaTa within the cell were usually accompanied withredistribution of AKT to the site of ZaTa. In addition to the cellularredistribution of AKT upon entry of ZaTa, there was also a decrease inthe phosphorylation level of AKT substrates in these cells. Similarresults were obtained in vivo after stereotactic injection offluorescent ZaTa into the frontal cortex, wherein a specific reductionin phosphorylation level of AKT substrates was observed in cells thatwere positive for ZaTa. These in vitro and in vivo observations showedthat ZaTa not only co-localized with AKT inside the cell but alsoinhibited its catalytic activity.

Entry of ZaTa into the cell also caused different patterns of expressionof PKAc, depending on the localization of ZaTa. For example, there was asignificant decrease in PKAc immunoreactivity in cells displaying astrong nuclear signal for ZaTa, whereas cells with cytoplasmicaggregates of ZaTa generally showed an increase in PKAc protein levels.These data indicate that the proper activity of AKT in the cell caninfluence the expression of PKAc. Not wishing to be bound by theory, itis believed that nuclear redistribution of AKT, due to treatment withZaTa, caused transcriptional changes that suppressed the expression ofPKAc. Alternatively, redistribution of ZaTa within cytoplasmiccompartments could have caused a compensatory effect, i.e., upregulationof PKAc, to compensate for the decrease in activity of AKT.

The phenotypic consequence of disrupting the AKT-PKAc complex was alsodetermined. ZaTa peptide was injected into the striatum of onehemisphere of the brain and DMSO, as vehicle, was injected into theother brain hemisphere of an adult C57BL/6 mouse under anesthesia. Thebrain was removed and dissected. Equal protein amounts from eachhemisphere were subjected to immunoprecipitation by an anti-AKTantibody. The amount of AKT protein immunoprecipitated from the right(vehicle-treated) and left (ZaTa-treated) striatum were comparable;however, the amount of PKAc in physical contact with AKT wasdramatically reduced after treatment with ZaTa. This showed that ZaTacould disrupt the physical complex between AKT and PKAc in vivo. Tocompare PKAc protein levels in the ZaTa- and vehicle-treated brainhemisphere lysates, western blot analysis was conducted. Although therewere comparable amounts of PKAc in both hemispheres, a clear increase inmolecular weight was observed for PKAc in the hemisphere treated withZaTa. This indicates that treatment with ZaTa caused an electro-mobilitychange in PKAc, possibly due to post-translational changes in PKAcmolecules.

As disclosed herein, N2a cells showed a neurite-specific pattern ofAKT-PKAc interaction on their neuronal cell processes. Accordingly, thestability and phenotypic consequences of the AKT-PKAc complex was alsoanalyzed in cultured neurons. N2a cells were treated with vehicle, ZaTaor control peptide for 24 hours. Media was removed after the 24-hourtreatment and an equal number of cells from each treatment group waseither cultured for another 24 hours without treatment, or lysed andsubjected to immunoprecipitation with an antibody against AKT. Thosecells cultured for another 24 hours were also harvested and subjected toimmunoprecipitation. In parallel, the number of individual neurons withneurites was counted. Treatment with ZaTa reduced the amount of PKAc inphysical contact with AKT, an effect which was reversible by a 24-hourincubation in the absence of ZaTa. Concurrently, cells treated with ZaTaexhibited a significant reduction in the number of neurons with neurites(FIG. 2), an effect which was reversible by a 24-hour incubation in theabsence of ZaTa. These data not only confirmed the in vivo observationsshowing the disruption of AKT-PKAc complex by ZaTa, but also showed thecorrelation of neurite formation with the amount of PKAc in physicalcontact with AKT in cultured neurons. Moreover, these data indicate thatthe effect of ZaTa is reversible. Live images of N2a neurons werecaptured following treatment with vehicle or ZaTa (2 or 5 μM). Theseimages showed the normal pattern of neurite morphology in untreated N2acells, wherein treatment of N2a cells with ZaTa peptide caused dramaticmorphological changes, in a dose-dependent manner. ZaTa-mediated changesincluded a progressive loss of neurites, inhibition of new neuriteformation, loss of cell motility, as well as formation of large cellcolonies.

To determine the phenotypic effect of disrupting the AKT-PKAc complex inan in vivo setting, ZaTa peptide was stereotactically injected into thebrain of a mouse and the animal was perfused 18 hours after recoveryfrom surgery. Because AKT is known to have a role in axonal morphology(Markus, et al. (2002) Neuron 35:65-76), coronal sections of striatumwere stained with neurofilament-H (NF-H), as an axonal-specific marker.Changes in the staining pattern of axonal filament bundles in striatumwere observed upon stereotactic injection of ZaTa peptide, as comparedto the other brain hemisphere injected with DMSO as vehicle. To rule outthe effect of tissue damage and show that striatal tissue structure wasmaintained following surgery, sections were co-labeled with a nuclearmarker (Draq5). NF-H and nuclear marker staining of the same Z stepshowed similar tissue structure in both vehicle- and ZaTa-treatedhemispheres of the same coronal section. These data, consistent with theco-localization observations disclosed herein, indicate a role for AKTin axon growth and the acceleration of axonal regeneration (see alsoMarkus, et al. (2002) supra; Namikawa, et al. (2000) J. Neurosci.20:2875-2886). PKA is also known to have a role in regeneration ofgrowth cones on axons (Chierzi, et al. (2005) Eur. J. Neurosci.21:2051-2062). Therefore, given the data provided herein, it is believedthat a proper interaction between AKT and PKAc is involved inmaintaining normal neuronal morphology.

AKT affects a network that positively regulates G1/S cell cycleprogression through several mechanisms that involve the expression andsubcellular localization of the CDK inhibitor p27^(Kip1) (Blain andMassague (2002) Nat. Med. 8:1076-1078; Liang, et al. (2002) supra; Shin,et al. (2002) supra; Viglietto, et al. (2002) supra). Based on thesestudies, the effect of ZaTa peptide on cell proliferation was assessed.Using an MTT-based proliferation assay, a dose-dependent decrease in thenumber of live cells was observed. Western blot analysis showed aconcomitant decrease in the phosphorylation level of AKT substrates inN2a cells after treatment with different doses of ZaTa peptide.Capturing live images of cultured N2a cells in the presence of differentconcentrations of the ZaTa peptide confirmed an obvious reduction in thenumber of dividing cells. Thus, consistent with the previous reportsshowing a positive role of AKT in cell cycle progression, the datadisclosed herein demonstrate the inhibitory role of the ZaTa peptide inthe rate of cell proliferation.

Based on the importance of the C-terminal arm of ZaTa for its inhibitoryaction on AKT, it was determined whether the free hydroxyl groups onresidues Thr-8, Thr-10 (equivalent to Thr-197 in full-length PKAc),Thr-14 and Tyr-17 were important for the biological activity of thispeptide. Mutants of ZaTa peptide were synthesized by replacing Thr-8 orThr-10 residues with Asp (ZaTa^(T8D) and ZaTa^(T10D), respectively) asan amino acid with a negatively charged side chain. Mutants of ZaTa withthe hydrophilic positively charged amino acid Arg at either positionThr-14 or Tyr-17 (ZaTa^(T14R) and ZaTa^(Y17R), respectively) were alsosynthesized. Replacing either Thr-8 or Thr-10 with an Asp significantlydiminished the inhibitory effect of ZaTa on cell proliferation, whilereplacing Thr-14 or Tyr-17 with an Arg considerably augmented ZaTaactivity (FIG. 3, histogram series 1). The inhibitory effect of ZaTapeptide on cell proliferation was reversible, to a large extent, afterthe removal of ZaTa treatment (FIG. 3, histogram series 2). Whilewild-type ZaTa caused a significant decrease in the number of live cellswith doses as low as 2 μM, no significant differences in the number oflive cells were observed 24 hours after removal of 10 μM wild-type ZaTaor DMSO vehicle. These observations showed that the biochemicalproperties of the side chain of the amino acids composing the primarystructure of ZaTa peptide were important for the biological effects ofthis peptide. It is possible that ZaTa, like other peptides, can switchbetween the alpha/beta secondary structures, with one structure morefavorable for its active conformation while the other one creates aninactive form. Therefore, the mutation of Thr-14 or Thr-17 to an Argappeared to stabilize the structure of ZaTa to its active conformationwhile changing Thr-8 or Thr-10 to Asp was more favorable for generationof an inactive conformation.

Some cell proliferation assays, such as MTT, do not distinguish whethera decrease in the number of viable cells is due to a decrease in thenumber of dividing cells, or is a result of cell toxicity and death.Therefore, in addition to capturing live images, cells were stained withtrypan blue at different time intervals following treatment with ZaTaand counted by a light microscope using a hemocytometer. The samedose-dependent decrease in the number of live cells was observed.However, although a significant increase in the number of dead cells wasnot seen after treatment with wild-type ZaTa, ZaTa^(T8D), orZaTa^(T10D), counted at different time points from 12 to 72 hours, asignificant increase in the number of dead cells was observed aftertreatment with ZaTa^(T14R) or ZaTa^(Y17R) only after 72 hours. Thisobservation indicated that while the inhibitory effect of the wild-typeZaTa on AKT was reversible, the ZaTa^(T14R) or ZaTa^(Y17R) mutantscould, by causing an irreversible inhibition of AKT, cause permanentchanges leading to apoptosis and cell death. Alternatively, it waspossible that the free hydroxyl group on Thr-14 or Tyr-17 created anunstable/cleavable binding of ZaTa with AKT, while the Arg-14 or Arg-17made this binding more stable and non-cleavable.

The interaction between PKAc and AKT at the transcriptional level wasalso evaluated by decreasing PKAc alpha protein levels by RNAinterference. Reduced PKAc levels resulted in an increase in the amountof AKT1 protein in non-neuronal HeLa cells as well as in neuronal NG-108cells. AKT expression was also analyzed in a PRKACA (PKAc alpha)knockout mouse. Since homozygous knockout mice of this strain do notsurvive to adulthood, AKT1 protein levels were measured in aheterozygous PKAc mouse, which expressed ˜50% of the PKAc proteincompared to wild-type. Protein extracts from the frontal cortex of theheterozygous PKAc mouse showed an increase in the AKT1 protein level.These data showed that in addition to the physical interaction betweenAKT and PKA, which affected their activity levels directly, there wereactive transcriptional mechanisms involved that regulated the proteinlevel as well.

It is now well-established that AKT protects against apoptosis throughphosphorylation and inhibition of pro-apoptotic mediators such as BAD,FOXO family members and IKK-β (Datta, et al. (1999) Genes Dev.13:2905-2927). To demonstrate the effect of ZaTa on the protectivefunction of AKT, non-proliferating neurons in primary cortical culturewere analyzed as a model system that utilizes the minimal level of thecell proliferative activity of AKT. Primary neurons were treated withDMSO, 1 μM or 5 μM of either ZaTa or the control peptide for a durationof 1, 3, 16, 24, 48 or 72 hours. The number of apoptotic cells wascounted following the TUNEL assay. The result of this experiment showeda marked dose-dependent increase in the number of TUNEL-positive cellsafter a 72-hour treatment with ZaTa as compared to the control peptide.The increase in the number of apoptotic neurons following treatment withZaTa was consistent with its potent intra-neuronal inhibition of AKT.

To confirm the apoptotic inducing effect of ZaTa in vivo, ZaTa wasdelivered to the mouse brain via the nasal cavity, a minimally invasiveprocedure compared to the stereotactic surgery. Intranasal delivery ofcompounds into the brain is an efficient and effective way for localdelivery of compounds, without the need for passing the blood brainbarriers, the major obstacle for studying the effect of differentinhibitors/activators in CNS (Vyas, et al. (2005) Curr. Drug Deliv.2:165-175; Hrafnkelsdottir, et al. (2005) Biol. Pharm. Bull.28:1038-1042). Repeated intranasal treatment of C57BL/6 mice withlabeled ZaTa for three days significantly increased the number ofapoptotic cells in the olfactory bulb, specifically in cells stainedpositive for ZaTa. This was visualized by double labeling of the brainsections with fluorescent TUNEL and a nuclear marker. By contrast, nochange in the number of apoptotic cells was observed in cells whichstained negative for ZaTa or following treatment with the controlpeptide. Taken together, these data indicate that ZaTa can inhibitAKT-dependent functions both in vitro and in vivo.

To improve in vitro activity, in vivo efficacy, and stability of ZaTa, aseries of modified peptides have been prepared. The present disclosuretherefore relates to compositions of ZaTa-modified peptides for use inmethods of changing kinase activity in the treatment of diseases orconditions associated with aberrant expression of AKT. The disclosedcompositions embraced by the present disclosure include pharmaceuticalcompositions containing the ZaTa-modified peptides in admixture with apharmaceutically acceptable carrier. As ZaTa and its mutants were foundto inhibit AKT activity and/or modify the activity of other kinases,particular embodiments embrace pharmaceutical compositions containingone or more of ZaTa analogs.

Rational design of the modified peptides is facilitated by the knowncrystal structure of an activated AKT in complex with GSK-3 peptide andAMP-PNP (Yang, et al. (2002) Nat. Struct. Biol. 9:940-944). Thestructure revealed the binding of GSK-3 peptide through the activationloop of AKT. The observation that the short sequence of ZaTa peptide(SEQ ID NO:60), surrounding the Thr-197 located in the activation loopof PKAc, was sufficient to inhibit AKT as potently as the full-lengthPKAc protein indicates that during the course of interaction between theactive conformations of the two molecules, residues adjacent to theThr-197 site are essential and sufficient for this inhibition. Notwishing to be bound by theory, it is believed that in the activeconformation of full-length PKAc, a specific sequence surroundingThr-197 docks into the active site of AKT thereby preventing efficientphosphorylation of Thr-308 and/or binding of GKS-3 substrate peptide tothe activation loop of AKT and AKT fails to phosphorylate GSK-3 at Ser-9site. Looking at the other component of this interaction, it is foundthat in contrast to the inhibitory effect of active PKAc, AKTphosphorylates PKAc at Thr-197 which increases its catalytic activity.The data disclosed herein indicate that this phosphorylation is notrequired for the inhibitory effect of PKAc toward AKT; however, itprovides a conformational change that not only favors a more activestate for PKAc, but also exposes residues surrounding this site for thesubsequent inhibitory effect of full-length PKAc on AKT. Therefore, as astructural model, the PKAc/AKT interaction functions as a molecularon/off switch in which AKT phosphorylates Thr-197 of PKAc first, whichresults in a more active conformation for PKAc and its binding to theactivation loop of AKT provides an inactive conformation for AKT. In ananalysis of cAMP-induced activation of PKA, the crystal structure of thecatalytic and regulatory (RIα) subunits of PKA in complex was determined(Kim, et al. (2005) Science 307:690-696). This analysis indicates thatthe PKA inhibitor peptide of the RI subunit is sufficient to inhibitPKAc catalytic activity.

A series of modifications were applied to the sequence of ZaTa peptideto identify the shortest active fragment of ZaTa peptide. Severalfragments from ZaTa peptide were synthesized and tested in-vitro by MTTassay to find the shortest sequence that can show anti-cellproliferative activities. This experiments revealed that a variant ofZaTa with the sequence as short as Lys-Gly-Arg-Thr-(1-Nal)-Thr-Leu-Cysis sufficient to inhibit cell proliferation.

Furthermore, the ability of the ZaTa peptide to form a dimer of itssingle Cys residue and the effect of this dimerization on the activitylevel and the potency of the ZaTa peptide were analyzed both in vitroand in vivo. For in-vitro testing, the anti-cell proliferative activityof the monomer of ZaTa was compared to the dimer for using an MTT assay.This experiment showed that the dimer has more potency for theinhibition of cell proliferation compared to the monomer of the peptide.In in vivo assays, the tumor growth rate of U251 xenografts werecompared between the animals that were treated with the vehicle, thedimer or the monomer of the ZaTa peptide. This animal study alsoconfirmed that the dimer form has better activities compared to themonomer.

Moreover, typical peptide modifications at different residues such aspegylation and lipidation of the peptide were tested and studied bothin-vitro and in-vivo. This study showed that both lipidation and/orpegylation of the peptide facilitate the anti-cell proliferativeactivity of the peptide both in vitro and in vivo.

Other changes, such as C terminal acylation and N-terminal amidation ofthe peptide also improved the anti-cell proliferative activity of ZaTapeptide. However, adding a fluorescent molecule such as FITC in order tovisualize the activity of the peptide seemed to reduce the anti-cellproliferative activities of ZaTa peptide. Significantly, replacing thehydrophilic residues of ZaTa, such as its Thr-15 or Tyr-17, withhalogenated amino acid derivatives such as 4-Cl Tyr or 4-F Tyr or4-Cl-Phe or 4-F-Phe improved the activity of ZaTa. Moreover, changes ofnatural amino-acids such as the Lys to a similar non-proteinogenic aminoacid such as Orn, also improved the activity of ZaTa.

Changes in the sequence of ZaTa peptide, such as single replacement ofresidues, or simultaneous replacement of two or three residues withamino-acids with similar properties in terms of hydrophobicity andpositive or negative or neutral charge, did not significantly changedthe interfere with the anti-cell proliferative peptide activity. Thisexperiments reflects the fact that the sequence of this peptide cantolerate several mutations in its sequence without blocking itsanti-cell proliferation activity.

Whether inhibition of AKT1 activity is required for the anti-cellproliferative activity of ZaTa was examined. Several variants of ZaTawere synthesized with different truncations and mutations and weretested during in-vitro kinase assays against AKT1. This study showedthat there in fact several variants of ZaTa that do not inhibit AKT1significantly, but can still inhibit cell proliferation. This experimentshowed that inhibition of AKT1 is one mechanism of action for ZaTa andthe anti-cell proliferative activity of ZaTa variants does not requireinhibition of AKT1.

Using in-vitro kinase assays, the kinase inhibition profiles of ZaTavariants were tested against ˜115 different kinases involved in cellproliferation. This kinase profiling showed that besides AKT1, AKT2,p70S6K and Abl, several other kinases can be inhibited in nano-molarrange, by different variants of ZaTa peptide. More specifically, thekinases that were inhibited within the nano-molar range of concentrationof different variants were:

-   -   AKT1 (PKB alpha), AKT2 (PKB beta), MAP3K8 (COT), MST4, AURKB        (Aurora B), ROCK1, RPS6KB1 (p70S6K), CDC42 BPA (MRCKA), BRAF,        RAF1 (cRAF) Y340D Y341D, SGK (SGK1), MAP4K4 (HGK), AURKA (Aurora        A), AURKC (Aurora C), BRAF V599E, CHEK1 (CHK1), GSG2 (Haspin),        CHEK2 (CHK2), FGR, IKBKB (IKK beta), CDK7/cyclin H/MNAT1, and        CDC42 BPB (MRCKB)

Accordingly, in one aspect, the present disclosure embraces a modifiedpeptide including:

-   -   (i) an amino acid sequence (X)-GRT-(Y)-TLC-(Z), or    -   (ii) an amino acid sequence having at least 40% sequence        identity to the amino acid sequence (X)-GRT-(Y)-TLC-(Z),    -   wherein    -   X is a natural amino acid, a non-natural amino acid, a chemical        modification of a natural or non-natural amino acid, an acetyl        group, a lipid group, or a combination thereof;    -   Y is a natural amino acid, a non-natural amino acid, a chemical        modification of a natural or non-natural amino acid, or a        combination thereof; and    -   Z is a natural amino acid, a non-natural amino acid, a chemical        modification of a natural or non-natural amino acid, an amine        group, or a combination thereof.

In any aspect or embodiment described herein, X may include an acetylgroup, a lauroyl group, or a palmitoyl group located at the terminal endthereof; Y may be 1-Nal, 2-Nal; and Z may include an amino group locatedat the terminal end thereof.

In any aspect or embodiment described herein, the amino acid sequencemay be (X¹)-KGRT-(Y)-TLC-(Z), wherein X¹ is the same as X above.

In any aspect or embodiment described herein, the amino acid sequencemay be (X²)-VKGRT-(Y)-TLC-(Z), wherein X² is the same as X above.

In any aspect or embodiment described herein, the amino acid sequencemay be (X³)-RVKGRT-(Y)-TLCGRPE-(Z¹), wherein X³ is the same as X above,and wherein Z¹ is the same as Z above.

In any aspect or embodiment described herein, the amino acid sequencemay be (X⁴)-KRVKGRT-(Y)-TLCGRPE-(Z¹), wherein X⁴ is the same as X above,and wherein Z¹ is the same as Z above.

In any aspect or embodiment described herein, the amino acid sequencemay be (X⁵)-RVKGRT-(Y)-TLCGRPE-(Z¹), wherein X⁵ is the same as X above,and wherein Z¹ is the same as Z above.

In any aspect or embodiment described herein, the amino acid sequencemay be (X⁷)-V-(X⁸)-GRT-(Y)-TLC-(Z), wherein X⁷ is the same as X above,and wherein X⁸ is a natural amino acid, a non-natural amino acid, achemical modification of a natural or non-natural amino acid, or acombination thereof.

In any aspect or embodiment described herein, the amino acid sequencemay be (X⁹)-KV-(X⁸)-GRT-(Y)-TLC-(Z), wherein X⁹ is the same as X above.

In another aspect, the present disclosure embraces a modified peptidehaving the formula:

(X)-(seq1)-(Y)-(seq2)-(Z) or an amino acid sequence having at least 40%sequence identity to the amino acid sequence (X)-(seq1)-(Y)-(seq2)-(Z)

wherein

seq1 is GRT, KGRT, VKGRT, RVKGRT, KRVKGRT, (Orn)-RVKGRT or AKRVKGRT;

seq2 is TLC, TLCG, TLCGR, TLCGRPE, TLCGRPEY or TLCGRPE-(4-Cl-Phe);

X is a natural amino acid, a non-natural amino acid, a chemicalmodification of a natural or non-natural amino acid, an acetyl group, alipid group, or a combination thereof;

Y is a natural amino acid, a non-natural amino acid, a chemicalmodification of a natural or non-natural amino acid, or a combinationthereof; and

Z is a natural amino acid, a non-natural amino acid, a chemicalmodification of a natural or non-natural amino acid, an amine group, ora combination thereof.

In any aspect or embodiment described herein, X may include an acetylgroup or a lipid group located at the terminal end thereof. The lipidgroup may be a C6 to C20 lipid group, for example, a lauroyl group or apalmitoyl group.

In any aspect or embodiment described herein, Y may be 1-Nal, and Z mayinclude an amino group located at its terminal end.

In any aspect or embodiment described herein, the non-natural amino acidis ornithine, naphthylalanine, 4-chloro phenylalanine, or a combinationthereof.

In another aspect, the present disclosure embraces a dimer of the abovemodified peptide. In any aspect or embodiment described herein, thedimer is a homodimer or a heterodimer, and may include a disulfide bond.

In accordance with the present disclosure, exemplary modified peptidesmay include the following structures:

Lauroyl-(Orn)-RVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH₂ (Cys-Cys dimer)Lauroyl-(Orn)-RVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH₂Palmitoyl-(Orn)-RVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH₂ (Cys-Cys dimer)Palmitoyl-(Orn)-RVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH₂Ac-(Orn)-RVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH₂ (Cys-Cys dimer)Ac-(Orn)-RVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH₂Ac-AKRVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH₂Ac-AKRVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH₂ (Cys-Cys dimer)Ac-KRVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH₂Ac-KRVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH₂ (Cys-Cys dimer)Ac-KGRT-(1-Nal)-TLC-NH₂ Ac-KGRT-(1-Nal)-TLC-NH₂ (Cys-Cys dimer)Ac-VKGRT-(1-Nal)-TLC-NH₂ Ac-VKGRT-(1-Nal)-TLC-NH₂ (Cys-Cys dimer)Ac-V-(Orn)-GRT-(1-Nal)-TLC-NH₂Ac-V-(Orn)-GRT-(1-Nal)-TLC-NH₂ (Cys-Cys dimer)Ac-V-(Orn)-GRT-(1-Nal)-TLCG-NH₂Ac-V-(Orn)-GRT-(1-Nal)-TLCG-NH₂ (Cys-Cys dimer)Ac-V-(Orn)-GRT-(1-Nall)-TLCGR-NH₂Ac-V-(Orn)-GRT-(1-Nal)-TLCGR-NH₂ (Cys-Cys dimer)Ac-(Orn)-GRT-(1-Nal)-TLC-(4-Cl-Phe)-NH₂Ac-(Orn)-GRT-(1-Nal)-TLC--(4-Cl-Phe)-NH₂ (Cys-Cys dimer)Ac-(Orn)-GRT-(1-Nal)-TLC-NH₂Ac-(Orn)-GRT-(1-Nal)-TLC-NH₂ (Cys-Cys dimer)Ac-KV-(Orn)-GRT-(1-Nal)-TLC-NH₂Ac-KV-(Orn)-GRT-(1-Nal)-TLC-NH₂ (Cys-Cys dimer)Ac-KVKGRT-(1-Nal)-TLC-NH₂ Ac-KVKGRT-(1-Nal)-TLC-NH₂ (Cys-Cys dimer)Ac-RVKGRT-(1-Nal)-TLC-NH₂ Ac-RVKGRT-(1-Nal)-TLC-NH₂ (Cys-Cys dimer).

In the above structures, “Lauroyl” is n-dodecanoyl, “Palmitoyl” isn-hexadecanoyl, “Ac” is acetyl, “Orn” is ornithine, “1-Nal” is1-naphthylalanine, “2-Nal” is 2-naphthylalanine, “4-Cl-Phe” is4-chloro-phenylalanine, and “Cys” is cysteine.

As used herein, “modified peptides” of the present disclosureencompasses polypeptides that are recombinantly produced, purified froma natural source, or chemically synthesized. For yield and ease inpurification, it is conventional in the art to produce proteins andfragments thereof by recombinant protein methodologies. Methods forproducing recombinant proteins in vivo (i.e., cell-based) generallyinclude isolating a nucleic acid molecule encoding the protein orfragment of interest, incorporating the nucleic acid molecule into arecombinant expression vector in a form suitable for expression of theprotein or fragment in a host cell, and expressing the protein. Asuitable form for expression provides that the recombinant expressionvector includes one or more regulatory sequences operatively-linked tothe nucleic acid molecule encoding the protein or fragment of interestin a manner which allows for transcription of the nucleic acids intomRNA and translation of the mRNA into the protein. Regulatory sequencescan include promoters, enhancers and other expression control elements(e.g., polyadenylation signals). Such regulatory sequences and vectorsencoding the same are known to those skilled in the art and aredescribed in Goeddel, Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. (1990). Suitable vectors forrecombinant protein expression in mammalian, yeast, or prokaryoticsystems are commercially available from such sources as STRATAGENE®,INVITROGEN™, Pharmacia and the like. Many of these vectors encodeheterologous polypeptides, i.e. signal sequences for secretion and/orother polypeptide which will aid in the purification of the protein orfragment of interest. Preferably, the heterologous polypeptide has aspecific cleavage site to remove the heterologous polypeptide from theprotein of interest. Other useful heterologous polypeptides which can befused to the protein of interest are those which increase expression orsolubility of the fusion protein or aid in the purification of thefusion protein by acting as a ligand in affinity purification. Typicalfusion expression vectors include pGEX (Amersham Biosciences,Piscataway, N.J.), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5(Pharmacia, Piscataway, N.J.), which fuse glutathione-S-transferase,maltose E binding protein, or protein A, respectively, to the protein ofinterest. It should be understood that the design of the expressionvector may depend on such factors as the choice of the host cell to betransfected and/or the level of expression required.

Introduction of the recombinant expression vector into a host cell(e.g., of eukaryotic or prokaryotic origin) can be carried out using anyconventional technique for transforming cells. Suitable methods fortransforming host cells are found in Sambrook, et al. (MolecularCloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor LaboratoryPress (2000)) and other laboratory manuals. The number of host cellstransformed with a nucleic acid molecule encoding a protein will depend,at least in part, upon the type of recombinant expression vector usedand the type of transformation technique used. A recombinant protein orfragment can be expressed transiently, or more typically, stablyexpressed by integrating the recombinant expression vector into thegenome of the host cell or by episomal maintenance of the vector.

Once produced, a modified peptide can be recovered from culture mediumas a secreted polypeptide, or alternatively recovered from host celllysates when directly expressed without a secretory signal. When amodified peptide is expressed in a recombinant host cell other than oneof human origin, the modified peptide is substantially free of proteinsor polypeptides of human origin. However, it may be necessary to purifythe modified peptide from recombinant cell proteins or polypeptidesusing conventional protein purification methods to obtain preparationsthat are substantially homogeneous as to the modified peptide. As afirst step, the culture medium or lysate is centrifuged to removeparticulate cell debris. The membrane and soluble protein fractions arethen separated. The recombinant modified peptide may then be purifiedfrom the soluble protein fraction. The recombinant modified peptidethereafter is purified from contaminant soluble proteins andpolypeptides using any of the following suitable purificationprocedures: by fractionation on immunoaffinity or ion-exchange columns;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; gel filtration using, for example,Sephadex™ G-75; and ligand affinity chromatography.

In addition to recombinant production, a modified peptide can beproduced by direct peptide synthesis using solid-phase techniques(Merrifield R. B. (1963) J. Am. Chem. Soc. 85:2149-2154). Proteinsynthesis can be performed using manual techniques or by automation.Automated synthesis can be achieved, for example, using AppliedBiosystems® 431A Peptide Synthesizer (Perkin Elmer, Boston, Mass.). Whenproducing a modified peptide, various portions thereof can bechemically-synthesized separately and combined using chemical methods toproduce a full-length molecule.

Whether recombinantly-produced or chemically-synthesized, a modifiedpeptide can be further functionalized for use. For example, a modifiedpeptide can be phosphorylated, acetylated, methylated or a combinationthereof using well-known methods in prior to its use in inhibiting theactivity of AKT and other kinases. Moreover, PKAc and PKAcfragment-based therapeutics can be attached to a modified peptidescaffold.

In any aspect or embodiment described herein, the amino acid residues inthe modified peptide of present disclosure are selected from any of thenaturally-occurring amino acids. In other embodiments, one or more orsynthetic non-encoded amino acids are used to replace one or more of thenaturally-occurring amino acid residues. Certain commonly encounterednon-encoded amino acids include, but are not limited to: peptidemimetics or analogs; beta or gamma amino acids; the D-enantiomers of thegenetically-encoded amino acids; 2,3-diaminopropionic acid (Dpr);α-aminoisobutyric acid (Aib); ε-aminohexanoic acid (Aha); δ-aminovalericacid (Ava); N-methylglycine or sarcosine (MeGly or Sar); ornithine(Orn); citrulline (Cit); t-butylalanine (Bua); t-butylglycine (Bug);N-methylisoleucine (MeIle); phenylglycine (Phg); cyclohexylalanine(Cha); norleucine (Nle); homoleucine (hLeu), homovaline (hVal);homoisolencine (hIle); homoarginine (hArg); N-acetyl lysine (AcLys);2,4-diaminobutyric acid (Dbu); 2,3-diaminobutyric acid (Dab);N-methylvaline (MeVal); homocysteine (hCys); homoserine (hSer);hydroxyproline (Hyp) and homoproline (hPro); and the like. Additionalnon-encoded amino acids are well-known to those of skill in the art(see, e.g., the various amino acids provided in Fasman (1989) CRCPractical Handbook of Biochemistry and Molecular Biology, CRC Press,Boca Raton, Fla., at pp. 3-70 and the references cited therein).Further, amino acids of the inventions of the present disclosure can bein either the L- or D-configuration.

A modified peptide can be used as a purified preparation, or in certainembodiments, formulated into a pharmaceutical composition containing aneffective amount of a modified peptide or its dimer to decrease theexpression or activity of AKT and/or other kinases. Such pharmaceuticalcompositions can be prepared by methods and contain carriers which arewell-known in the art. A generally recognized compendium of such methodsand ingredients is Remington: The Science and Practice of Pharmacy,Alfonso R. Gennaro, editor, 20th ed. Lippincott Williams & Wilkins:Philadelphia, Pa., 2000. For example, sterile saline andphosphate-buffered saline at physiological pH can be used.Preservatives, stabilizers, dyes and even flavoring agents can beincluded in the pharmaceutical composition. For example, sodiumbenzoate, sorbic acid and esters of p-hydroxybenzoic acid can be addedas preservatives. In addition, antioxidants and suspending agents can beused. Liposomes, such as those described in U.S. Pat. No. 5,422,120, WO95/13796, WO 91/14445, or EP 524,968 B1, are also suitable carriers.

Depending on the intended use, a pharmaceutical composition of thepresent disclosure can be administered by any suitable means, includingparenteral injection (such as intraperitoneal, subcutaneous,intratumoral or intramuscular injection), orally or by topicalapplication (e.g., transdermal or via a mucosal surface). Bypharmaceutically acceptable formulation is meant, a composition orformulation that allows for the effective distribution of the peptidemolecules of the present disclosure in the physical location mostsuitable for their desired activity. Non-limiting examples of agentssuitable for formulation with the peptide molecules of the presentdisclosure include: PEG conjugated nucleic acids, phospholipidconjugated nucleic acids, nucleic acids containing lipophilic moieties,phosphorothioates, P-glycoprotein inhibitors (such as Pluronic P85)which can enhance entry of drugs into various tissues, for example theCNS (Jolliet-Riant and Tillement, 1999, Fundam. Clin. Pharmacol., 13,16-26); biodegradable polymers, such as poly (DL-lactide-coglycolide)microspheres for sustained release delivery after implantation (Emerich,D F et al, 1999, Cell Transplant, 8, 47-58) Alkermes, Inc. Cambridge,Mass.; and loaded nanoparticles, such as those made ofpolybutylcyanoacrylate, which can deliver drugs across the blood brainbarrier and can alter neuronal uptake mechanisms (ProgNeuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999). Othernon-limiting examples of delivery strategies, including CNS delivery ofinclude material described in Boado et al., 1998, J. Pharm. Sci., 87,1308-1315; Tyler et al, 1999, FEBS Lett., 421, 280-284; Pardridge etal., 1995, PNAS USA, 92, 5592-5596; Boado, 1995, Adv. Drug DeliveryRev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26,4910-4916; and Tyler et al., 1999, PNAS USA, 96, 7053-7058. All thesereferences are hereby incorporated herein by reference. The modifiedpeptides of the present disclosure were delivered into the brain viaintranasal injection; the peptides were dissolved in tetraglycol (Sigma)to 0.5 mM final concentration, and 5 μl of the solution was injected toeach nasal cavity.

In certain embodiments, the disclosure provides modified peptides thatare cell penetrating peptides and distribute rapidly throughout thehuman tissue. In certain embodiments, the peptides described herein aredelivered locally into the site of tumor. In an exemplary embodiment,the modified peptides of the present disclosure are injected directlyinto the site of the tumor through, for example, stereotactic surgery.However, one potential disadvantage of this technique is that localinjections are not generally formulated for sustained release delivery.

Therefore, additional formulation/delivery devices are also contemplatedthat provide for and/or are adapted for controlled and/or sustainedrelease of a therapeutic of the present disclosure. For example, themodified peptides of the present disclosure can be conjugated (eithercovalently or via non-covalent bonds) or merely entrapped in apharmaceutically acceptable (i.e., biologically inert or biologicallycompatible) and/or biologically absorbable carrier material, for examplea polymer matrix, biopolymer matrix, and/or other matrix. As usedherein, “biologically inert or biologically compatible” refers tomaterials that do not result in a significant allergic or immunogenicreaction in the host. In an embodiment, the material is comprised ofcollagen. Other materials include proteins, like elastin, saccharidesand gels, and/or sols comprising saccharides, for example, hydroxypropylcellulose (HPC), HPMC, methacrylates, and the like. In an exemplaryembodiment, the material is an absorbable collagen sponge (ACS) orcross-linked collagen matrix, which is adapted to allow controlledand/or sustained release of the peptide into the tissue. The modifiedpeptide could be inserted into a device or preshaped/prefabricatedmatrix material either contemporaneously or after formation of thedelivery device. In still other embodiments the modifiedpeptide/biocompatible material (e.g., collagen) could be inserted intoanother device, which is also bioabsorbable and/or implantable, thedevice to be delivered into the tumor site to allow sustained localdelivery. The combination of modified peptide/biocompatible materialcould also be inserted through a different external device. See, McKay,B. Local Sustained Delivery of Recombinant Human Bone MorphogeneticProtein-2 (rhHBMP-2). 31^(st) Annual International Conference of theIEEE EMBS, Sep. 2-6, 2009; and Chan, B. P. Effects of PhotochemicalCross-linking on the Microstructure of Collagen and a Feasability Studyon Controlled Protein Release. Acta Biomaterialia, 4:1627-36 (2008),which are hereby incorporated by reference in their entirety.

The formulations can be administered orally, topically, parenterally, byinhalation or spray or rectally in dosage unit formulations containingconventional non-toxic pharmaceutically acceptable carriers, adjuvantsand vehicles. The term parenteral as used herein includes percutaneous,subcutaneous, intravascular (e.g., intravenous), intramuscular, orintrathecal injection or infusion techniques and the like. In addition,there is provided a pharmaceutical formulation comprising a nucleic acidmolecule of the present disclosure and a pharmaceutically acceptablecarrier. One or more nucleic acid molecules of the present disclosurecan be present in association with one or more non-toxicpharmaceutically acceptable carriers and/or diluents and/or adjuvants,and if desired other active ingredients. The pharmaceutical compositionsof the present disclosure can be in a form suitable for oral use, forexample, as tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules, emulsion, hard or soft capsules, orsyrups or elixirs.

Compositions intended for oral use can be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions can contain one or more suchsweetening agents, flavoring agents, coloring agents or preservativeagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients can be for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia, and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques. In some cases such coatings can beprepared by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents can beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents orsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, can also be present.

Pharmaceutical compositions of the present disclosure can also be in theform of oil-in-water emulsions. The oily phase can be a vegetable oil ora mineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations can also contain a demulcent, a preservative and flavoringand coloring agents. The pharmaceutical compositions can be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilcan be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

Peptide molecules of the present disclosure can also be administered inthe form of suppositories, e.g., for rectal administration of the drugor via a catheter directly to the bladder itself. These compositions canbe prepared by mixing the drug with a suitable non-irritating excipientthat is solid at ordinary temperatures but liquid at the rectaltemperature and will therefore melt in the rectum to release the drug.Such materials include cocoa butter and polyethylene glycols.

Peptide molecules of the present disclosure can be administeredparenterally in a sterile medium. The drug, depending on the vehicle andconcentration used, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anesthetics, preservatives andbuffering agents can be dissolved in the vehicle.

The amount of active ingredient that can be combined with the carriermaterials to produce a single dosage form varies depending upon the hosttreated and the particular mode of administration. Dosage unit formsgenerally contain between from about 0.1 mg to about 1000 mg of anactive ingredient.

It is understood that the specific dose level for any particular patientor subject depends upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,sex, diet, time of administration, route of administration, and rate ofexcretion, drug combination and the severity of the particular diseaseundergoing therapy.

For administration to non-human animals, the composition can also beadded to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It can also be convenient to presentthe composition as a premix for addition to the feed or drinking water.

The composition can also be administered to a subject in combinationwith other therapeutic compounds to increase the overall therapeuticeffect. The use of multiple compounds to treat an indication canincrease the beneficial effects while reducing the presence of sideeffects.

In certain embodiments, the present disclosure encompasses host cellsthat have been modified to carry an exogenous or heterologous nucleicacid comprising a nucleic acid encoding for the modified peptide.

The term “host cell” includes a cell that might be used to carry aheterologous nucleic acid, or expresses a peptide or protein encoded bya heterologous nucleic acid. A host cell can contain genes that are notfound within the native (non-recombinant) form of the cell, genes foundin the native form of the cell where the genes are modified andre-introduced into the cell by artificial means, or a nucleic acidendogenous to the cell that has been artificially modified withoutremoving the nucleic acid from the cell. A host cell may be eukaryoticor prokaryotic. General growth conditions necessary for the culture ofbacteria can be found in texts such as BERGEY'S MANUAL OF SYSTEMATICBACTERIOLOGY, Vol. 1, N. R. Krieg, ed., Williams and Wilkins,Baltimore/London (1984). A “host cell” can also be one in which theendogenous genes or promoters or both have been modified to produce oneor more of the polypeptide components of the complex of the presentdisclosure.

Derivatives or variants of the nucleic acids, proteins, or peptides ofthe present disclosure include, but are not limited to, moleculescomprising regions that are substantially homologous to the nucleicacids or proteins of the present disclosure, in various embodiments, byat least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% identity (witha preferred identity of 80-95%) over a nucleic acid or amino acidsequence of identical size or when compared to an aligned sequence inwhich the alignment is done by a computer homology program known in theart, or whose encoding nucleic acid is capable of hybridizing to thecomplement of a sequence encoding the proteins of the present disclosureunder stringent, moderately stringent, or low stringent conditions. See,e.g., Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, JohnWiley & Sons, New York, N.Y., 1993. Nucleic acid derivatives andmodifications include those obtained by gene replacement, site-specificmutation, deletion, insertion, recombination, repair, shuffling,endonuclease digestion, PCR, subcloning, and related techniques.

Furthermore, one of ordinary skill will recognize that “conservativemutations” also include the substitution, deletion or addition ofnucleic acids that alter, add or delete a single amino acid or a smallnumber of amino acids in a coding sequence where the nucleic acidalterations result in the substitution of a chemically similar aminoacid. Amino acids that may serve as conservative substitutions for eachother include the following: Basic: Arginine (R), Lysine (K), Histidine(H); Acidic: Aspartic acid (D), Glutamic acid (E), Asparagine (N),Glutamine (Q); hydrophilic: Glycine (G), Alanine (A), Valine (V),Leucine (L), Isoleucine (I); Hydrophobic: Phenylalanine (F), Tyrosine(Y), Tryptophan (W); Sulfur-containing: Methionine (M), Cysteine (C). Inaddition, sequences that differ by conservative variations are generallyhomologous.

Descriptions of the molecular biological techniques useful to thepractice of the present disclosure including mutagenesis, PCR, cloning,and the like include Berger and Kimmel, GUIDE TO MOLECULAR CLONINGTECHNIQUES, METHODS IN ENZYMOLOGY, volume 152, Academic Press, Inc., SanDiego, Calif. (Berger); Sambrook et al., MOLECULAR CLONING--A LABORATORYMANUAL (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989, and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, F. M.Ausubel et al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc.; Berger,Sambrook, and Ausubel, as well as Mullis et al., U.S. Pat. No. 4,683,202(1987); PCR PROTOCOLS A GUIDE TO METHODS AND APPLICATIONS (Innis et al.eds), Academic Press, Inc., San Diego, Calif. (1990) (Innis); Arnheim &Levinson (Oct. 1, 1990) C&EN 36-47.

In yet another embodiment, a nucleic acid of the present disclosure isexpressed in mammalian cells using a mammalian expression vector. Forsuitable expression systems for both prokaryotic and eukaryotic cellssee, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: ALABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

A polynucleotide can be a DNA molecule, a cDNA molecule, genomic DNAmolecule, or an RNA molecule. A polynucleotide as DNA or RNA can includea sequence wherein T (thymidine) can also be U (uracil). If a nucleotideat a certain position of a polynucleotide is capable of forming aWatson-Crick pairing with a nucleotide at the same position in ananti-parallel DNA or RNA strand, then the polynucleotide and the DNA orRNA molecule are complementary to each other at that position. Thepolynucleotide and the DNA or RNA molecule are substantiallycomplementary to each other when a sufficient number of correspondingpositions in each molecule are occupied by nucleotides that canhybridize with each other in order to effect the desired process.

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques as are well known to those skilled in the art.By “transformation” is meant a permanent or transient genetic changeinduced in a cell following incorporation of new DNA (i.e., DNAexogenous to the cell).

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert, et al.,1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame andEaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of Tcell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) andimmunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen andBaltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci.USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985.Science 230: 912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990.Science 249: 374-379) and the alpha-fetoprotein promoter (Campes andTilghman, 1989. Genes Dev. 3: 537-546).

In any of the embodiments, the nucleic acids encoding the modifiedpeptides of the present disclosure can be present as: one or more nakedDNAs; one or more nucleic acids disposed in an appropriate expressionvector and maintained episomally; one or more nucleic acids incorporatedinto the host cell's genome; a modified version of an endogenous geneencoding the components of the complex; one or more nucleic acids incombination with one or more regulatory nucleic acid sequences; orcombinations thereof. The nucleic acid may optionally comprise a linkerpeptide or fusion protein component, for example, His-Tag, FLAG-Tag,fluorescent protein, GST, TAT, an antibody portion, a signal peptide,and the like, at the 5′ end, the 3′ end, or at any location within theORF.

Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂) methodby procedures well known in the art. Alternatively, MgCl₂, RbCl,liposome, or liposome-protein conjugate can be used. Transformation canalso be performed after forming a protoplast of the host cell or byelectroporation. These examples are not limiting on the presentdisclosure; numerous techniques exist for transfecting host cells thatare well known by those of skill in the art and which are contemplatedas being within the scope of the present disclosure.

When the host is a eukaryote, such methods of transfection with DNAinclude calcium phosphate co-precipitates, conventional mechanicalprocedures such as microinjection, electroporation, insertion of aplasmid encased in liposomes, or virus vectors, as well as others knownin the art, may be used. The eukaryotic cell may be a yeast cell (e.g.,Saccharomyces cerevisiae) or may be a mammalian cell, including a humancell. For long-term, high-yield production of recombinant proteins,stable expression is preferred.

As exemplified herein, the modified peptides of the present disclosurefind application in inhibiting the expression or activity of AKT and/orother kinases (e.g., as determined by phosphorylation of the Ser-21GSK-3 substrate peptide), wherein kinase inhibition results in adecrease in cell proliferation and a progressive dose-dependent loss ofthe existing neurites, as well as the inhibition of new neuriteformation. Accordingly, not only does the present disclosure embrace theuse of the modified peptides for decreasing proliferation of a cell, thepresent disclosure provides methods for preventing or treating cancer ora neurodegenerative or psychiatric disease or condition.

AKT-mediated control of cell cycle progression is well-established inthe art (see, e.g., Brazil, et al. (2004) supra). AKT regulates the cellcycle by facilitating G1/S transition and the initiation of M phase(Collado, et al. (2000) J. Biol. Chem. 275:21960-21968; Datta, et al.(1999) Genes Dev. 13:2905-2927; Franke, et al. (1997) Cell 88:435-437).AKT also phosphorylates MDM2 which causes its translocation to thenucleus, where it promotes the degradation of p53, leading to areduction in the transcription of p21^(Cip1) mRNA. In the nucleus, FOXOtranscription factors increase the transcription of p27^(Kip1), but thisfunction is inhibited by AKT phosphorylation, which causes FOXO proteinsto remain in the cytoplasm. The cyclin-dependent kinase (CDK) inhibitorp21^(Cip1) and p27^(Kip1) proteins can also be phosphorylated by AKT,leading to their accumulation in the cytoplasm, which relieves theinhibition of CDK2 activity and facilitates G1/S transition (Blain andMassague (2002) supra; Liang, et al. (2002) supra; Shin, et al. (2002)supra; Viglietto, et al. (2002) supra). AKT also drives the cell cycleto M phase by phosphorylating a checkpoint protein with FHA and ringfinger domains (CHFR) and Myt1 (Brazil, et al. (2004) supra; Okumura, etal. (2002) supra). In view of the fact that AKT plays an important rolein regulation of multiple checkpoints during the cell cycle, andhyperactivity of AKT is known to be involved in the most prevalent humanmalignancies including breast cancer, prostate cancer, lung cancer,gastrointestinal tumors, pancreatic cancer, hepatocellular carcinoma,thyroid cancer and CNS malignancies (such as glioblastoma and gliomas),the modified peptides of the present disclosure can be used forinhibiting cancer cell proliferation, e.g., in the prevention andtreatment of cancer.

Glioblastoma is the most common primary central nervous system tumor inadults. Mitotic activity in glioblastoma is abundant, and vascularendothelial proliferation is prominent. Both of these two mechanisms aretightly regulated by AKT through phosphorylation and protein-proteininteraction (Brazil, et al. (2002) supra). These features cause a rapidgrowth rate and most patients die within one year of diagnosis(Underwood (2004) General and systemic pathology. 4^(th) Edition). AKTsignaling pathway is implicated in tumor initiation and maintenance ofglioblastoma and gliomas (Lefranc, et al. (2005) J. Clin. Oncol.23:2411-22; Kesari, et al. (2005) Curr. Neurol. Neurosci. Rep. 5:186-97)and targeting AKT is an effective strategy for treating of brain tumors(Kesari, et al. (2005) supra). Inhibitors of AKT have been investigatedin clinical trials for treatment of glioblastoma (Carpentier (2005)Bull. Cancer 92:355-9). The effect of monoclonal antibodies and smallpeptidic hormones for local targeting of malignant gliomas has beeninvestigated (Merlo, et al. (2003) Acta Neurochir. Suppl. 88:83-91) withsignificant tumor uptake by small peptidic hormone receptors.

The exemplary modified peptides disclosed herein were found to beeffectively absorbed and distributed throughout mouse brain tissue andspecifically inhibit AKT following local administration of a very smalldose of this peptide (only 1 μL of mM solution). The modified peptidesalso potently inhibit cell proliferation of cancerous cells derived fromdifferent malignant human cell lines. Considering the combination of thethree effects of the modified peptides, i.e., distribution in braintissue, inhibition of AKT and other peptides in vivo, and inhibition ofcell proliferation, the modified peptides will be useful in treatment ofhuman CNS tumors, as well as a number of other human malignancies, inwhich these three processes have been shown to play an important role inpathology development and poor prognosis. In treatment of CNS tumors,the modified peptides of the present disclosure have the advantage ofbeing delivered directly into the tumor site using advanced andminimally invasive neurosurgical techniques. Current treatments of CNStumors usually involve either invasive neurosurgery with potentialserious post-surgical complications or intensive radiotherapy.

Activation of the AKT pathway has also been demonstrated to contributeto the pathogenesis of prostate cancer (Culig, et al. (2005) Endocr.Relat. Cancer 12:229-44), and inhibition of this signaling pathway isknown to have therapeutic implications in human prostate adenocarcinoma(Wang, et al. (2004) Neuron 38:915-928). Therefore, targeting AKT withthe modified peptides of the present disclosure can be used in treatmentof prostate cancer as well.

AKT is also documented as being involved in breast cancer. Whilepeptide-based vaccines are commonly used for targeting breast cancer(Disis, et al. (2004) Breast Dis. 20:3-11), the modified peptides of thepresent disclosure can be used as primary or adjunct therapeutic agentsin the treatment of breast cancer.

A method for inhibiting cell proliferation generally involves the stepof contacting a cell (e.g., a cancer cell) with an effective amount ofthe modified peptide (e.g., in a pharmaceutical composition), therebyreducing the proliferation of the cell as compared to a cell notcontacted with the modified peptide. Means for monitoring the reductionof cell proliferation are disclosed herein.

In the context of cancer cell proliferation and cancer prevention ortreatment, an effective amount is considered an amount that decreases orinhibits cancer cell proliferation such that tumor development isarrested and/or tumor size is reduced. Desirably, the agent causes a10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% decreasein cancer cell proliferation or tumor size when compared to otherwisesame conditions wherein the modified peptide is not present.

As used herein, “effective amount” is used to refer to the amount of themodified peptide required to prevent, inhibit the occurrence, or treat(alleviate a symptom to some extent, preferably all of the symptoms) ofa disease state. The effective dose depends on the type of disease, thecomposition used, the route of administration, the type of animal beingtreated, the physical characteristics of the specific animal underconsideration (e.g., age, weight, gender), concurrent medication, andother factors which those skilled in the medical arts will recognize.Generally, an amount between 0.001 mg/kg and 1000 mg/kg body weight/dayof active ingredients is administered dependent upon potency. Theinventions of the present disclosure includes pharmaceuticalcompositions that include therapeutically- or prophylactically-effectiveamounts of a therapeutic and a pharmaceutically-acceptable excipient.

In an embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationsfor the dosage unit forms of the present disclosure are dictated by anddirectly dependent on the unique characteristics of the active compoundand the particular therapeutic effect to be achieved, and thelimitations inherent in the art of compounding such an active compoundfor the treatment of individuals.

The nucleic acid molecules of the present disclosure can be insertedinto vectors and used as gene therapy vectors. Gene therapy vectors canbe delivered to a subject by, for example, intravenous injection, localadministration (see, e.g., U.S. Pat. No. 5,328,470) or by stereotacticinjection (see, e.g., Chen, et al., 1994. Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells that producethe gene delivery system. The pharmaceutical compositions can beincluded in a container, pack, or dispenser together with instructionsfor administration.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds that exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects. The data obtainedfrom the cell culture assays and animal studies can be used informulating a range of dosage for use in humans. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED₅₀ with little or no toxicity. The dosage may varywithin this range depending upon the dosage form employed and the routeof administration utilized. For any compound used in the method of thepresent disclosure, the therapeutically effective dose can be estimatedinitially from cell culture assays. A dose may be formulated in animalmodels to achieve a circulating plasma concentration range that includesthe IC₅₀ (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

In so far as the instant compositions decrease or inhibit cancer cellproliferation, individuals having or at risk of having a cancer such asbreast cancer, prostate cancer, lung cancer, gastrointestinal tumors,pancreatic cancer, hepatocellular carcinoma, thyroid cancer or CNSmalignancies (such as glioblastoma and gliomas) would benefit byreceiving treatment with a the modified peptide of the presentdisclosure or a composition including the same. Individuals havingcancer generally refer to patients who have been diagnosed with cancer,whereas individuals at risk of having cancer may have a family historyof cancer or exhibit one or more signs or symptoms associated withcancer (e.g., a tumor, increased pain perception, weakness). Suchindividuals, upon receiving treatment with a composition of the presentdisclosure, are expected to exhibit a decrease in the signs or symptomsassociated with cancer and a general improvement in the quality of lifeand life expectancy. It is contemplated that not only will the instantcompositions be useful in the prevention or treatment of malignancies,said compositions will also find application in the treatment of benigntumors, e.g., benign CNS tumors. While benign CNS tumors do notmetastasize, they can cause significant complications and disabilitiesas the result of their high growth tendency in the skull and puttingpressure on the important CNS structures. Thus, treatment with theinstant compositions would provide relief from such symptoms.

Given the enhanced cell targeting activity of a modified peptide of thepresent disclosure, particular embodiments embrace the use of themodified peptide as a moiety for targeted delivery of a therapeutic orcontrast agent to a cell or tissue. As such, the instant modifiedpeptide can be operatively linked, e.g., via a covalent attachment, to achemotherapy or therapeutic agent to increase cellular targeting anduptake of the agent as compared to the unconjugated agent.Alternatively, the modified peptide can be attached to the surface of adrug-loaded liposome or nanoparticle for facilitating delivery of drugto a cell. Agents which can be targeted to a cell (e.g., a cancer cellor neuron) using the modified peptides of the present disclosure includecytotoxic agents such as Taxol, Cytochalasin B, Gramicidin D, EthidiumBromide, Emetine, Mitomycin, Etoposide, Tenoposide, Vincristine,Vinblastine, camptothecin (CPT), Colchicin, Doxorubicin, Daunorubicin,Mitoxantrone, Mithramycin, Actinomycin D, 1-Dehydrotestosterone,Glucocorticoids, Procaine, Tetracaine, Lidocaine, Propranolol, andPuromycin; therapeutic agents including antimetabolites (e.g.,Methotrexate, 6-Mercaptopurine, 6-Thioguanine, Cytarabine,5-Fluorouracil, Decarbazine), alkylating agents (e.g., Mechlorethamine,Thiotepa, Chlorambucil, Melphalan, Carmustine (BCNU), Lomustine (CCNU),Cyclophosphamide, Busulfan, Dibromomannitol, Streptozotocin, MitomycinC, Cis-Dichlorodiamine Platinum (II) (DDP), Cisplatin), anthracyclines(e.g., Daunorubicin (formerly Daunomycin) and Doxorubicin), antibiotics(e.g., Dactinomycin (formerly Actinomycin), Bleomycin, Mithramycin, andAnthramycin (AMC)), anti-inflammatory agents, anti-mitotic agents (e.g.,Vincristine and Vinblastine) and selective apoptotic agents such asAPTOSYN® (Exisulind), PANZEM™ (2-methoxyestradiol), VELCADE®(bortezomib) a proteasome inhibitor, cytotoxic agents, alkylating agent,antimetabolite, anthracycline, plant alkaloid, topoisomerase inhibitor,antibody, kinase inhibitor, or other antitumor agents, radioisotopes,therapeutic nucleic acids or polypeptides, fluorescent markers,paramagnetic ions, contrast reagents, metal chelators, toxins, hormonessuch as steroids; antimetabolites such as cytosine arabinoside,fluorouracil, methotrexate or aminopterin; anthracycline; mitomycin C;vinca alkaloids; demecolcine; etopo side; mithramycin; or alkylatingagents such as chlorambucil or melphalan, chemotherapeutic agents, suchas antitumor drugs, nucleic acids, nucleotides, cytokines,antimetabolites, alkylating agents, antineoplastic agents, peptide orpseudopeptide chelating agents (e.g., linker-chelator,glycyl-tyrosyl-(N, e-diethylenetriaminepentaacetic-acid)-lysinehydrochloride (GYK-DTPA-HCl), radioactive compounds, diphtheria toxin(chain A), ricin toxin (chain A), adriamycin, chlorambucil,daunorubicin, or pokeweed antiviral protein to enhance their tumoricidaleffectiveness, nuclear magnetic spin-resonance isotopes, metallic ions,and the like. However, as would be understood by those of skill in theart, the present disclosure is not limited to any particular type orclass of therapeutic agent, or any particular disease to be treated.

Methods for performing conjugation of the agents listed above to apeptide or pseudopeptide are well known or readily determinable, andinclude, for example, conjugation to amino acid side chains, functionalgroups, carbohydrates, lipids, and other small molecules. See forexample, Goldenberg, D. M. et al, New England J. Med., 298:1384-1388(1978), Goldenberg, D. M. et al, J. A. M. A., 250:630-635 (1983),Goldenberg. D. M. et al, Gastroenterol., 84:524-532 (1983), Siccardi, A.G. et al, Cancer Res., 46:4817-4822 (1986). Epenetos, A. A. et al,Cancer, 55:984-987 (1985), Philben, V. J. et al, Cancer, 57:571-576(1986), Chiou, R. et al, Cancer Res., 45:6140-6146 (1985) and Hwang, K.M. et al, J. Natl. Cancer Inst., 76:849-855 (1986), all of which arespecifically incorporated herein by reference.

Examples of markers which can be conjugated to the antibody are wellknown to those skilled in the art and include substances which can bedetected by nuclear magnetic resonance imaging, i.e., nuclear magneticspin-resonance isotopes, and radioactive substances. A preferred exampleof a nuclear magnetic spin-resonance isotope is gadolinium (Gd).Suitable examples of radioactive markers include I¹²⁵, I¹³¹, I¹²³,In¹¹¹, In¹¹³, Ga⁶⁷, Ga⁶⁸, Ru⁹⁷, Ru¹⁰³, Hg¹⁹⁷, Hg²⁰³, and Tc⁹⁹. Detectionof radioactive markers is by means of a gamma scintillation camera orthe like as described in the references cited above. Nuclear magneticimaging devices can be used to detect nuclear magnetic spin-resonanceisotope markers.

In general, a modified peptide of the present disclosure has anamphipathic nature with a net positive charge. Generally, amphipathicstructures play an important role in mediating the interaction ofpeptides and proteins with membranes (Sharadadevi, et al. (2005)Proteins 59:791-801). Because primary amphipathic cell penetratingpeptides have been used for the efficient intracellular delivery oflarge hydrophilic molecules such as oligonucleotides and proteins, theyhave been used in drug delivery (Plenat, et al. (2005) Biophys. J.89:4300-4309). It has been shown that in amphipathic helices there is astrong preference for Arg or Lys to occur (Sharadadevi, et al. (2005)supra). There is also a relationship between the net charge and averagehydrophobic moments, the determining factor for the membrane seekingproperties. A net positive charge appears to favor higher hydrophobicmoment than a net negative charge (Sharadadevi, et al. (2005) supra).Like known cell penetrating peptides, the amphipathic structure of themodified peptide can facilitate penetration into the cell, targeting AKTand PKAc and other kinases inside the cell and exerting its biologicalactivity. Mutations of Thr-14 or Tyr-17 to Arg generates a higher netpositive charge thereby increasing the average hydrophobic moments forthe modified peptide and augmenting its effect, whereas the mutations ofThr-8 or 10 to the negatively charged Asp causes a less positive netcharge, thereby moderating biological activity. Accordingly, themodified peptides of the present disclosure are useful not only asanticancer agents, but also for targeted delivery of an additionaltherapeutic agents to a cell.

Several other peptide modifications in the sequence of ZaTa improved itsactivity. Examples of such modification are replacing single, two orthree amino-acids, replacing natural amino-acids with similar unusualones, such as changing Try to Nal, or Lys to Orn, or replacing thenatural hydrophilic residues such as Thr or Tyr to halogenated aminoacid derivatives such as 4-Cl Tyr or 4-F Tyr or 4-Cl-Phe or 4-F-Phe.

As in cancer, an increase in the activity of AKT is also known to beassociated with different types of Alzheimer's disease (Blain andMassague, (2002) supra; Griffin, et al. (2005) supra; Liang, et al.(2002) supra; Shin, et al. (2002) supra; Viglietto, et al. (2002)supra), wherein reduced AKT activity is related to schizophrenia(Emamian, et al. (2004) supra). Moreover, the PKA signaling pathway hasa novel role for in schizophrenia as well (Millar, et al. (2005) supra).

Regarding neuronal synapse activity and neurodegeneration, ion channelshave been identified as a novel class of PKB/Akt substrates, pinpointingsynaptic plasticity as a biological process regulated by this kinase. Inparticular, the β2 subunit of the type A γ-aminobutyric acid (GABA_(A))receptor is an AKT substrate in vitro and in vivo (Wang, et al. (2002)Neuron 38:915-928). This protein is a member of a ligand-gated chlorideion channel that mediates synaptic transmission at most inhibitorysynapses in the mammalian brain. Drugs such as benzodiazapines andbarbiturates act on the GABA_(A) receptor to mediate anti-psychoticeffects. AKT-mediated phosphorylation of Ser-410 increases the number ofGABA_(A) receptors on the plasma membrane surface, thereby increasingthe efficacy of receptor-mediated inhibition at GABAergic synapses(Brazil, et al. (2004) supra).

Studies in humans provide evidence of increased AKT activation andhyperphosphorylation of critical AKT substrates in Alzheimer's disease(AD) brain (Griffin, et al. (2005) supra). Differential distribution ofAKT and phospho-AKT is observed in AD temporal cortex neurons comparedwith control neurons, which is accompanied with increased levels ofactive phosphorylated-AKT in particulate fractions, and significantdecreases in AKT levels in AD cytosolic fractions, causing increasedactivation of AKT (phosphorylated-AKT/total AKT ratio) in AD. Further,significant increases in the levels of phosphorylation of total AKTsubstrates, including GSK3β(Ser-9), tau(Ser-214), mTOR(Ser-2448), anddecreased levels of the AKT target, p27^(kip1), is reported in ADtemporal cortex compared with controls. Moreover, a significant loss andaltered distribution of the major negative regulator of AKT, PTEN(phosphatase and tensin homologue deleted on chromosome 10), is found inAD neurons. Loss of phosphorylated-AKT and PTEN-containing neurons isobserved in hippocampal CA1 at the end stages of AD (Griffin, et al.(2005) supra). Enzymatic activity of AKT in mid-temporal and mid-frontalcortices from AD cases and matched controls has also been analyzed(Rickle, et al. (2004) Neuroreport 15:955-959). The results of thisanalysis indicated that the activity of AKT (GSK-3α/β fusion proteinphosphorylation by immunoprecipitated AKT) was significantly increasedin temporal cortex soluble fractions from AD compared with non-diseasecontrols and positive disease controls with another neurodegenerativedisease. Moreover, AKT activity in temporal cortex soluble fractions waspositively correlated with Braak staging for neurofibrillary changes.Strong phospho-AKT immunoreactivity was shown in AD pyramidal neuronsundergoing degeneration and in reactive astroglia. Given that PKAcfragments can potently decrease AKT substrate phosphorylation in thebrain, inhibition of AKT could reverse the observed changes in humanswith AD thereby providing therapeutic benefit in the treatment of AD.

Many inherited neurodegenerative diseases are caused by the expansion ofa CAG repeat that produces a long polyglutamine (polyQ) tract inproteins, the length of which is directly correlated with the severityof the disease (Emamian, et al. (2003) Neuron 38:375-387; Chen, et al(2003) Cell 113:457-68). AKT substrates that mediate the pathophysiologyof spinocerebellar ataxia type 1 (SCA1) and Huntington's disease havebeen identified (Humbert, et al. (2002) Dev. Cell 2:831-837; Emamian, etal. (2003) supra; Chen, et al. (2003) supra). Toxicity of the mutantproteins in vivo is directly mediated by phosphorylation of Ser-776(Emamian, et al. (2003) supra). Replacing Ser-776 with Ala completelyaverts the pathology in vivo, even in the presence of a longpolyglutamine tract. Therefore, while polyglutamine expansion isrequired for the disease to develop, it is not sufficient. Based on thisanalysis, Ser-776 was identified as a site of AKT phosphorylation, amolecular event that is essential for the interaction of 14-3-3 with thepolyQ-expanded form ataxin-1 (Chen, et al. (2003) supra), whereinbinding to 14-3-3 triggers the formation of inclusion bodies ofataxin-1, mediating its neurotoxicity. In this regard, PKB/Akt-mediatedphosphorylation of the mutant form of ataxin-1 in SCA1 triggers 14-3-3binding, gradual accumulation of this protein, and consequentneurodegeneration.

Similar to SCA1, Huntington's disease is characterized by an expandedpolyQ repeat in the huntingtin protein, which leads to an aggregation ofmutated protein in the nucleus and selective apoptosis of striatalneurons in the brain (Saudou, et al. (1998) Cell 95:55-66). In contrastto its role in SCA1, however, AKT positively regulates the survival ofstriatal neurons lost during the degeneration seen in Huntington'sdisease. Both insulin-like growth factor 1 (IGF-1) treatment and AKTactivation of striatal neurons inhibits cell death and intranuclearinclusion formation mediated by the mutated huntingtin protein (Humbert,et al. (2002) supra). Phosphorylation of the mutated form of huntingtinby PKB/Akt on Ser-421 is required for IGF-1-mediated inhibition ofintranuclear inclusion formation and cell death, indicating thatcompromised AKT activity could accentuate the progression ofHuntington's disease. In this regard, analysis of AKT protein in brainsamples from individuals affected with Huntington's disease reveals thepresence of both full-length AKT (60 kDa) and a shorter form (49 kDa)predicted to be generated by caspase-3-mediated cleavage of thefull-length kinase (Humbert, et al. (2002) supra).

AKT signaling in neurons of amyotrophic lateral sclerosis has also beendetermined (Kaspar, et al. (2003) supra). IGF-1 stimulates the activityof PKB/Akt in the spinal cord and prolongs the lifespan of SOD1 mice byincreasing the survival of motor neurons in this setting, indicatingthat administration of IGF-1 could be of benefit in the treatment ofamyotrophic lateral sclerosis (Kaspar, et al. (2003) supra).

Direct evidence has been provided for the role of AKT in axonal growthand the acceleration of axonal regeneration (Markus, et al. (2002)supra; Namikawa, et al. (2000) Nat. Cell Biol. 4:111-116). Furthermore,PKA is also shown to play a role in the axonal pathfinding of zebrafisholfactory sensory neurons (Yoshida, et al. (2002) J. Neurosci.22:4964-4972), as well as the ability of axons to regenerate theirgrowth cones (Chierzi, et al. (2005) supra). Having demonstrated thatendogenous PKAc co-localizes with AKT in N2a neurons along the neuritelength, as well as in the neurite outgrowth zone, and treatment with themodified peptides of the present disclosure results in a progressivedose-dependent loss of the existing neurites, as well as the inhibitionof new neurite formation, methods for modulating neurodegenerative andpsychiatric diseases and conditions is also embraced by the presentdisclosure. In particular, based on the role of AKT in the pathogenesisof SCA1, Huntington's disease, ALS and AD, the modified peptides andtheir compositions are useful in the prevention and treatment of theseneurodegenerative diseases.

In another aspect, the present disclosure also provides a method forpreventing or treating an immunodeficiency disorder, e.g., AIDS, usingthe modified peptides (e.g., in a pharmaceutical composition). As aprophylactic or therapeutic, an effective amount of the instantcomposition is administered to a patient having (e.g., showing signs orsymptoms of disease) or at risk of having (e.g., geneticallypredisposed) an immunodeficiency disorder, e.g., AIDS, to prevent (i.e.,inhibit or delay the development or onset of) or treat (i.e., amelioratethe signs or symptoms of) the disease or disorder. Immunodeficiencydiseases or conditions embraced by the present disclosure include, butare not limited to, AIDS, leukemia, lymphoma, viral diseases, e.g.,hepatitis, multiple myeloma, ataxia-telangiectasia, Chediak-Higashisyndrome, combined immunodeficiency disease, complement deficiencies,DiGeorge syndrome, hypogammaglobulinemia, Job syndrome, leukocyteadhesion defects, panhypogammaglobulinemia, Bruton's disease, congenitalagammaglobulinemia, selective deficiency of IgA, and Wiskott-Aldrichsyndrome.

In another aspect, the present disclosure also provides a method forpreventing or treating a neurodegenerative disease or disorder using themodified peptides (e.g., in a pharmaceutical composition). As aprophylactic or therapeutic, an effective amount of the instantcomposition is administered to a patient having (e.g., showing signs orsymptoms of disease) or at risk of having (e.g., geneticallypredisposed) a neurodegenerative disease or disorder to prevent (i.e.,inhibit or delay the development or onset of) or treat (i.e., amelioratethe signs or symptoms of) the disease or disorder. Neurodegenerativediseases or conditions embraced by the present disclosure include, butare not limited to, SCA1, Huntington's disease, ALS and AD.

Large-scale gene therapy clinical trials for treatment of Parkinsondisease are known (Howard (2003) Nat. Biotechnol. 21(10):1117-8). Inthese trials, a gene is cloned into a recombinant expression vector thatis known to be deregulated in the disease and is delivered locally tothe site of pathology in the brain. Accordingly, it is contemplated thatthese gene therapy approaches in clinical trials make it possible to usethe same settings for the delivery of DNA molecules encoding PKAcproteins or fragments into the site of pathology. This approach can beused to overexpress PKAc proteins or fragments in tumor cells therebypreventing further division and growth, and eventually, resulting inapoptosis and death. As another example, expression of PKAc proteins orfragments in Purkinje cells of the cerebellum using a Purkinjecell-specific promoter (such as PCP-2) and an adenoviral vector system,could be used to inhibit AKT and the phosphorylation of ataxin-1 therebyinhibiting the binding of ataxin-1 with 14-3-3 proteins. As a result,further progression of the pathology is prevented by blocking anupstream critical signal that is required for the development ofpathology. As AKT knock out mice do not exhibit a cerebellar dysfunctionphenotype, inhibition of AKT in Purkinje cells is not expected to causeside effects, since AKT does not seem to have a crucial role in normalfunction of cerebellum.

The inventions of the present disclosure is described in greater detailby the following non-limiting examples.

Examples

In the examples below, the exemplary compound(s) and/or exemplarycomposition(s) refer to some compounds of Table 1, as well as all otherexemplary compounds described in the present disclosure, andcompositions comprising the same.

Analysis of the in vitro activity of the Exemplary compounds: Theexemplary compositions were selected among several variants of ZaTapeptide with significant modifications to improve its activity. TheExemplary compounds are cell penetrating peptides that passes thebarrier of cell membrane, can inhibit multiple important kinases in cellproliferation (including but not limited to AKT, Abl and P70S6K), caninduce apoptosis, and can potently inhibit cell proliferation. A seriesof in vitro assays to confirm that the activities of the Exemplarycompounds was performed. More specifically, the following were tested:the ability of each Exemplary compound to enter into the cell, thekinase inhibitory profile of each Exemplary compound by in-vitro andcell-based kinase assays, its effect on the induction of apoptosis, aswell as its potency for the inhibition of cell proliferation. Moreimportantly, the efficacy of the exemplary compounds was tested onanimal models of glioblastoma (GBM), and analyzed the toxicity,stability and solubility profiles using a number of in-vitro and rodentstudies. The studies described herein confirmed that the activities ofthe Exemplary compounds are well preserved and highly improved duringthe lead optimization process. More importantly, the studies proved thatthe Exemplary compounds are significantly superior to the existing drugsin terms of efficacy and toxicity profiles.

Inhibition of the kinase activity by the Exemplary compound: To test themulti-kinase inhibitory profile of the Exemplary compounds usingin-vitro kinase assays, the kinase inhibition profiles of the exemplarycompounds were tested against ˜115 different kinases involved in cellproliferation. This kinase profiling showed that besides AKT1, AKT2,p70S6K and Abl, several other kinases can be inhibited in nano-molarrange, by different variants of these compounds. More specifically, thekinases that were inhibited within the nano-molar range of concentrationof different compounds were: AKT1 (PKB alpha), AKT2 (PKB beta), MAP3K8(COT), MST4, AURKB (Aurora B), ROCK1, RPS6KB1 (p70S6K), CDC42 BPA(MRCKA), BRAF, RAF1 (cRAF) Y340D Y341D, SGK (SGK1), MAP4K4 (HGK), AURKA(Aurora A), AURKC (Aurora C), BRAF V599E, CHEK1 (CHK1), GSG2 (Haspin),CHEK2 (CHK2), FGR, IKBKB (IKK beta), CDK7/cyclin H/MNAT1, and CDC42 BPB(MRCKB). A cell-based kinase assay was designed to check the moleculartargets of the exemplary compounds in human cancer cells. U251 humanglioblastoma cells were treated with the exemplary compounds at severalconcentrations, and with controls, for different time intervals of 20min, 2 hours and 24 hours and the phosphorylation or total proteinlevels of several substrates were measured at a known phosphorylationsite for each kinase. Moreover, the total protein levels of fewpotential intracellular targets of a selected lead was also measured. Adecrease in the phosphorylation level of each substrate at a knownphosphorylation site would reflect the inhibition of the kinase activityby the Exemplary compound. FIG. 1 shows representative immunoblots fromcell-based assays by probing with antibodies that recognize a PI3K-P110,or phospho-PDK1 (Ser-241), total AKT1, phospho-P53 (Ser-46),phospho-AKT1 (Thr-308), p-CRAF (Ser-259), phospho-Aurora A (Thr-288), ortotal Aurora A. Cells based assays after treating the U251 humanglioblastoma cells with different concentrations of vehicle, or anExemplary compound from 5 uM to 40 uM at different time intervals of 30min, 2 hours or 24 hours, confirmed some of the targets of the exemplarycompounds by in-vitro kinase profiling and showed that the Exemplarycompounds are capable of targeting several kinases and inhibit theiractivities inside the cells as well in. This experiment also showed thatp53 is also downstream target of the Exemplary compounds (FIG. 1).

Cell penetration activity of the Exemplary compounds: Several cancercell lines were tested to study the cell penetration capabilities of themodified exemplary compounds. This experiment showed that the modifiedpeptides are still capable of entering into the cells. In U251 cells forexample, after 2 hours of treatment with a fluorescently labeled form ofthe Exemplary compound, ˜10-15% of the cells stain positively with theExemplary compound (images not shown). After 3 days of treatment withthe Exemplary compound this percentage significantly increases to 90%.The result of this experiment shows the stability of the cellpenetration property of the Exemplary compound during the optimizationprocess.

Induction of apoptosis by the Exemplary compound: An important aspect ofthe Exemplary compounds' activity is their ability to induce apoptosisin malignant cancer cells. The ability of the Exemplary compounds weretested to make sure the new modifications preserved the apoptosisinducing ability of the peptide. U251 human glioblastoma cells weretreated with the exemplary compounds for a few time intervals. FIG. 2shows the apoptosis and survival of U251 glioblastoma cells at differenttime intervals following the treatment with an Exemplary compounds.Equal number of U251 human glioblastoma cells (5×10⁵) were plated oncoverslips and treated with either vehicle (top panel) or with anExemplary compound (bottom panels) for 20 min, one, two or three days.Fluorescent TUNEL (green) assay was performed to visualize the apoptoticcells and DAPI staining was used to visualize the nuclei of all cells(blue). Indirect immunofluorescent staining of Cleaved-Caspase 3 (red)was performed as another marker of apoptosis. Cells treated with thevehicle grew to a much higher confluency compared to those treated withthe Exemplary compound (compared the DAPI signal of the top and bottompanels). The confocal image (20×) is the same Z step showing threechannels separately (the first three columns) and the merged image (lastcolumn). The images in FIG. 2 show a time dependent increase inapoptosis rate of the cells after treatment, after 3 days of treatmentalmost 100% of cells are apoptotic shown by both Tunel (green) andincreased Cleaved-Caspase 3 (red).

Quantitative analysis of the cell density and apoptosis rate atdifferent time intervals: Equal cell numbers were plated on coverslipsand were treated with the Exemplary compounds and vehicle for differenttime intervals. Cells were stained with DAPI and Tunel and analyzed withconfocal microscope. FIG. 3(A) shows histogram bars reflecting theaverage number of total cells left on coverslips (DAPI positive cells)counted on five separate confocal fields. FIG. 3 (B) is the histogrambars showing the average percent of apoptotic cells calculated bydividing the number of Tunel positive cells to the total cells left oncoverslips (DAPI positive cells) counted on five separate confocalfields. Cells treated with the vehicle constantly showed a higherdensity over time that reached to approximately full confluency at 72hours (not shown), while cells treated with the Exemplary compoundsconsistently showed a significant decrease in the cell density over timeand a time-dependent increase in the number of apoptotic cells, to theextent that almost 100% of the cells were apoptotic after 72 hours oftreatment (FIG. 3).

Inhibition of cell proliferation by the Exemplary compounds: The mostimportant activity of the Exemplary compounds are inhibition of cellproliferation. Therefore, the effect of three Exemplary compounds on thecell proliferation rate of several cancer cell types was measured. FIG.4 shows the IC50s of the Exemplary compounds in human breast, lung,blood and skin cancer cells. A representative plate of few human cancercell lines are shown in this figure, including the metastatic humanbreast cancer cells (BT-549 and MDA-MB-468), human acute promyelocyticleukemia (HL-60), a Multiple Myeloma cancer cell line (RPMI-8226), asmall-cell lung cancer cell line (DMS-114), a human melanoma cancer cellline (SK-ML-5). Cell were treated for three days with three Exemplarycompounds, the vehicle (PBS), or controls of other kinase inhibitorcompounds in clinical trial (MK-2206), or in clinical use (Imatinib orGleevec), serially diluted from 50 μM to 0.7 μM. This experiments showsthe superior efficacy and a nano-molar range of IC-50 of the Exemplarycompounds compared to the existing class of drugs in the market or inlatest stages of the clinical trials (FIG. 4). MTT cell proliferationassay were also performed to test the anti-cell proliferative activitiesof the Exemplary compounds and the Promega Cell Titer-Glo® assay tomeasure the IC-50 on several GBM cell lines. The IC-50 of the Exemplarycompounds on six different glioblastoma cells, including U87, U251,SNB-75, SF-268, SF-295, and SF-539, was between 0.2 to 2.5 μM, andbetween 0.6 to 1.2 μM on N2a neuroblastoma cells. This IC-50 is an orderof magnitude better than the IC-50 of Temodar® on the same cell lines(Sun et al., 2012, Torres et al., 2011, Milano et al., 2009, Kanzawa etal., 2004). The IC-50 of Temodar®, a leading brain tumor drug currentlyin the market, is 421-1021 μM on glioblastoma cells and 602 μM onNeuroblastoma cells (Sun et al., 2012, Tones et al., 2011, Milano etal., 2009, Kanzawa et al., 2004). The IC-50 reported for Gleevec is15.7-18.7 μM and 9-13 μM on glioblastoma and neuroblastoma cell lines,respectively (Beppu et al., 2004, Kinsella et al., 2011, Kinsella etal., 2012).

Comparative efficacy studies with few FDA approved multiple-kinaseinhibitors: To compare the efficacy of the Exemplary compound with otherFDA approved multi-kinase inhibitor drugs in clinical use, the IC50 ofthe Exemplary compounds was measured on human cells lines of livercancer (HCC), pancreatic cancer, and metastatic breast cancer, andcompared it to the IC50 of Sorafenib (approved for HCC), Lapatinib(approved for breast cancer), Sunitinib and Erlotinib (approved forpancreatic cancer). The data showed a highly superior efficacy andsignificantly lower IC50s of the Exemplary compounds compared toSorafenib, Sunitinib, Erlotinib and Lapatinib for the same FDA approvedindications.

Inhibition of tumor growth by the Exemplary compounds in animal models:To test the efficacy of the Exemplary compounds in animal models, and totest its ability to suppress tumor growth locally, flank xenografts ofglioblastoma (GBM), hepatocellular carcinoma (HCC), and metastaticbreast cancer were generated in female Nu/Nu mice of ˜6 weeks of age.Once tumors reached the volume of ˜100 mm³, animals were randomlydivided to two groups of vehicle vs. the compound treated, approximatelyeight animals per each group. Tumor sizes were measured by caliper,before the injections took place, at indicated time intervals followingthe injections, and shown in FIG. 5A for GBM, FIG. 5B for HCC, and FIG.5C for the metastatic breast cancer. During the trials, animals wereclosely monitored in terms of the weight loss and any signs of toxicity.Every three days, animals were administered a single dose of 50 mg/kg,in a 100 ul volume, of the Exemplary compound or 100 ul of saline,intratumorally. The results of the trial are presented in FIG. 5.Animals tolerated the dose of 50 mg/kg very well, without any weightloss or any visible signs of toxicity. The tumors in animals thatreceived the vehicle continued to grow very fast, but the tumors in Leadtreated group did not grow and were visibly much smaller than thecontrol group, as early as day 7 of the trial. In fact the tumors almostcompletely stopped growing and there was no need to increase the initialdose of the Exemplary compounds. Once tumors in the control groupreached the volume of ˜3000 mm³, all animals were sacrificed and thetumors were dissected out (FIG. 5). While the animals in the controlgroup looked severely cachectic and ill due to the advanced cancer, theanimals in the Exemplary compounds treated group looked healthy anddidn't have any signs of toxicity, and their weights were stable. Ahighly significant difference in tumor volumes was observed between thecontrol and the treated, as early as day seven after the firstinjection, which continued to be highly significant throughout the trial(FIGS. 5A, 5B, and 5C).

The results of the animal trials were particularly interesting whencompared to similar animal efficacy studies of other successful kinaseinhibitors that are either in the clinical use, such as Erlotinib (tradename Tarceva), or Lapatinib (trade names Tykerb and Tyverb), or thosethat are in Phase III of clinical trials, such as MK-2206 (an AKTinhibitor). Erlotinib is a receptor tyrosine kinase inhibitor, whichacts on the epidermal growth factor receptor (EGFR), and Lapatinib is adual tyrosine kinase inhibitor which interrupts the HER2/neu andepidermal growth factor receptor (EGFR) pathways. The study of Hirai etal has compared the efficacies of MK-2206, Erlotinib, and Lapatinib,either as single compound individually, or in combination therapy withone another, in xenograft models. Comparing the result of the trials ofthe Exemplary compound with the results of the efficacy of MK-2206,Erlotinib, and Lapatinib in the study by Hirai et al proves the highlysuperior efficacy of the Exemplary compounds compared to thesesuccessful drugs in a number of ways:

a) The Exemplary compounds show superior efficacy as a singlecomposition, none of the three MK-2206, Erlotinib, and Lapatinib showsimilar efficacy when are given individually.

b) The efficacy of the Exemplary compounds alone is even better than thecombination of MK-2206 and Erlotinib, or the combination of MK-2206 andLapatinib. This is an expected outcome since the Exemplary compoundstarget few critical kinases in cell proliferation simultaneously.

c) The effective dose of the Exemplary compound (50 mg/kg/three days) insame animals is much better than the effective dose of MK-2206 (60, 120and 360 mg/kg/day, Erlotinib (50 mg/kg/day), and Lapatinib (100mg/kg/day). The animal trial on the Exemplary compound was started atthe minimal dose of 50 mg/kg every three days and this minimal dose wassufficient to fully disrupt tumor growth.

Stability studies in vitro and in cell lines: The stability of theExemplary compositions were studied after incubation at differenttemperatures, including −20° C., 4° C., 25° C. and 40° C., and followingseveral cycles of freeze-thaw, in our non-GLP lab. Following theincubations several samples were drown and visibly examined for physicalsigns of instability, such as aggregation or precipitation. Samples werealso tested in GBM cell lines to see if the IC-50 has changed followingthe incubation (similar to the experiment presented in FIG. 4). Thisstudy confirmed the stability of the powder form of the Lead compositionfor several years at −20° C., for at least one year at 4° C., and forsix month at 25° C., and at least twelve week at 40° C. It alsotolerated up to ten cycles of freeze-thaw. This was no surprise becausethe sequence of the peptides are short and does not have any of theamino-acids that cause instability (residues such as Asn or Gln, whichare prone to deamidation, or Asp or Trp that are prone to hydrolysis andisomerization, or any free Cys to cause dimerization).

Solubility studies: Due to its amphipathic nature, the Exemplarycompositions are highly water soluble, it can be dissolved easily in anywater-based solution up to the concentration of 50 mg/ml. They can alsodissolve well in a number of organic solutions typically used in theformulation of pharmaceuticals.

Toxicity studies in vitro and in animals: As the first critical test fordrug development, the toxicity of the Exemplary compounds was analyzed,both in vitro and in rodents, and compared the results with severalother successful kinase inhibitor drugs. These test proved a superiortoxicity profile of the Exemplary compounds, compared to several otherkinase inhibitor drugs currently in the clinical use.

As the first in-vitro test, the toxicity of the Exemplary compounds wascompared with Gleevec, using a 98 well format of the Ames test inbacteria. The Ames test, which is one of the most frequently appliedtests in toxicology, is a Salmonella typhimurium reverse mutation assayidentification of carcinogens using mutagenicity in bacteria as anendpoint. Almost all new pharmaceutical substances and chemicals used inindustry are tested by this assay. The result of this test revealed thatthe Exemplary compounds are safe at several doses tested in bacteria. Asthe control assays, positive controls and negative controls and the samedoses of Gleevec were tested to validate the accuracy of this test.

As another commonly used in vitro toxicity test, hERG assay wasutilized. Numerous structurally and functionally unrelated drugs blockthe hERG potassium channel. hERG channels are involved in cardiac actionpotential repolarization, and reduced function of hERG lengthensventricular action potentials, prolongs the QT interval in anelectrocardiogram, and increases the risk for potentially fatalventricular arrhythmias. In order to reduce the risk of investingresources in a drug candidate that fails preclinical safety studiesbecause of QT prolongation, it is important to screen compounds foractivity on hERG channels early in the lead optimization process. TheIC-50 of the Exemplary compounds was measured against the hERG channelsusing the Predictor™ hERG Fluorescence Polarization Assay. This assayshowed that the Exemplary compounds do not inhibit hERG at any testedconcentrations.

The toxicity of the Exemplary compounds in rodents was also tested bymeasuring the Maximum Tolerated Dose (MTD) in mice and compared the dosewith the successful kinase inhibitors drugs in the market. Thesingle-dose MTD in C57BL/6 mice was determined for a range of dosesstarting from 25 mg/kg up to 400 mg/kg, with clinical score and weightas endpoints. Animals received a single dose of the Exemplary compoundsor Gleevec daily, started at 25 mg/kg on day one, 50 mg/kg on day 3, 100mg/kg in day 5, 200 mg/kg on day 7, and 400 mg/kg on day 9. Using thisdose escalation study, the Exemplary compounds were very well toleratedup to the dose 200 mg/kg, and Gleevec was well tolerated up to the doseof 100 mg/kg. A number of studies that have measured the toxicity ofseveral other chemotherapeutics in the clinic in the C57BL/6 mice (Astonet al., 2017) confirm that this dose is an acceptable MTD in C57BL/6mice. Comparing this dose with Gleevec's MTD, and also considering thefact that the chronic use of the Exemplary compounds at a single dose of50 mg/kg/3 days in Nu/Nu mice was well tolerated and completelydisrupted tumor growth, the Exemplary compounds will should pass thetoxicity in the Phase I clinical trial.

While this disclosure has been described in connection with what ispresently considered to be practical exemplary embodiments, it is to beunderstood that the inventions of the present disclosure is not limitedto the disclosed embodiments, but, on the contrary, is intended to covervarious modifications and equivalent arrangements included within thespirit and scope of the appended claims.

What is claimed is:
 1. A modified peptide having the formula:(X)-(seq1)-(Y)-(seq2)-(Z) or an amino acid sequence having at least 40%sequence identity to the amino acid sequence (X)-(seq1)-(Y)-(seq2)-(Z),wherein: seq1 is GRT, KGRT, VKGRT, RVKGRT, KRVKGRT, (Orn)-RVKGRT orAKRVKGRT; seq2 is TLC, TLCG, TLCGR, TLCGRPE, TLCGRPEY, orTLCGRPE-(4-Cl-Phe); X is a natural amino acid, a non-natural amino acid,a chemical modification of a natural or non-natural amino acid, anacetyl group, a lipid group, or a combination thereof; Y is a naturalamino acid, a non-natural amino acid, a chemical modification of anatural or non-natural amino acid, or a combination thereof; and Z is anatural amino acid, a non-natural amino acid, a chemical modification ofa natural or non-natural amino acid, an amine group, or a combinationthereof.
 2. The modified peptide according to claim 1, wherein themodified peptide comprises: (i) an amino acid sequence(X)-GRT-(Y)-TLC-(Z), or (ii) an amino acid sequence having at least 40%sequence identity to the amino acid sequence (X)-GRT-(Y)-TLC-(Z).
 3. Themodified peptide of claim 1, wherein the amino acid sequence is:(X¹)-KGRT-(Y)-TLC-(Z),

wherein X¹ is a natural amino acid, a non-natural amino acid, a chemicalmodification of a natural or non-natural amino acid, an acetyl group, alipid group, or a combination thereof.
 4. The modified peptide accordingto claim 1, wherein the amino acid sequence is: (X²)-VKGRT-(Y)-TLC-(Z),

wherein X² is a natural amino acid, a non-natural amino acid, a chemicalmodification of a natural or non-natural amino acid, an acetyl group, alipid group, or a combination thereof.
 5. The modified peptide accordingto claim 1, wherein the amino acid sequence is:(X³)-RVKGRT-(Y)-TLCGRPE-(Z¹),

wherein X³ is a natural amino acid, a non-natural amino acid, a chemicalmodification of a natural or non-natural amino acid, an acetyl group, alipid group, or a combination thereof, and wherein Z¹ is a natural aminoacid, a non-natural amino acid, a chemical modification of a natural ornon-natural amino acid, an amine group, or a combination thereof.
 6. Themodified peptide according to claim 1, wherein the amino acid sequenceis: (X⁴)-KRVKGRT-(Y)-TLCGRPE-(Z¹),

wherein X⁴ is a natural amino acid, a non-natural amino acid, a chemicalmodification of a natural or non-natural amino acid, an acetyl group, alipid group, or a combination thereof, and wherein Z¹ is a natural aminoacid, a non-natural amino acid, a chemical modification of a natural ornon-natural amino acid, an amine group, or a combination thereof.
 7. Themodified peptide according to claim 1, wherein the amino acid sequenceis: (X⁵)-RVKGRT-(Y)-TLCGRPE-(Z¹),

wherein X⁵ is a natural amino acid, a non-natural amino acid, a chemicalmodification of a natural or non-natural amino acid, an acetyl group, alipid group, or a combination thereof, and wherein Z¹ is a natural aminoacid, a non-natural amino acid, a chemical modification of a natural ornon-natural amino acid, an amine group, or a combination thereof.
 8. Themodified peptide according to claim 1, wherein the amino acid sequenceis: (X⁷)-V-(X⁸)-GRT-(Y)-TLC-(Z),

wherein X⁷ is a natural amino acid, a non-natural amino acid, a chemicalmodification of a natural or non-natural amino acid, an acetyl group, alipid group, or a combination thereof, and wherein X⁸ is a natural aminoacid, a non-natural amino acid, a chemical modification of a natural ornon-natural amino acid, or a combination thereof.
 9. The modifiedpeptide according to claim 1, wherein the amino acid sequence is:(X⁹)-KV-(X⁸)-GRT-(Y)-TLC-(Z),

wherein X⁹ is a natural amino acid, a non-natural amino acid, a chemicalmodification of a natural or non-natural amino acid, an acetyl group, alipid group, or a combination thereof, and wherein X⁸ is a natural aminoacid, a non-natural amino acid, a chemical modification of a natural ornon-natural amino acid, or a combination thereof.
 10. The modifiedpeptide according to claim 1, wherein: X comprises an acetyl group, alauroyl group, or a palmitoyl group located at the terminal end thereof;Y is 1-Nal; and Z comprises an amino group located at the terminal endthereof.
 11. The modified peptide according to claim 1, wherein at leastone of: X comprises an acetyl group or a lipid group located at theterminal end thereof; the lipid group is a C6 to C20 lipid group; Y is1-Nal; and Z comprises an amino group located at the terminal endthereof.
 12. The modified peptide according to claim 1, wherein thelipid group is a lauroyl group or a palmitoyl group.
 13. The modifiedpeptide according to claim 1, wherein the non-natural amino acid isornithine, naphthylalanine, 4-chloro phenylalanine, or a combinationthereof.
 14. A dimer of the modified peptide according to claim
 1. 15.The dimer according to claim 14, wherein the dimer comprises a disulfidebond.
 16. The dimer according to claim 14, wherein the dimer is ahomodimer or a heterodimer.
 17. The modified peptide according to claim1, wherein the modified peptide has the following formula: (i) thecompositions of the following structure:Lauroyl-(Orn)-RVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH₂ (Cys-Cys dimer),Lauroyl-(Orn)-RVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH₂,Palmitoyl-(Orn)-RVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH₂ (Cys-Cys dimer),Palmitoyl-(Orn)-RVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH₂,Ac-(Orn)-RVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH₂ (Cys-Cys dimer),Ac-(Orn)-RVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH₂,Ac-AKRVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH₂,Ac-AKRVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH₂ (Cys-Cys dimer),Ac-KRVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH₂,Ac-KRVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH₂ (Cys-Cys dimer),Ac-KGRT-(1-Nal)-TLC-NH₂, Ac-KGRT-(1-Nal)-TLC-NH₂ (Cys-Cys dimer),Ac-VKGRT-(1-Nal)-TLC-NH₂, Ac-VKGRT-(1-Nal)-TLC-NH₂ (Cys-Cys dimer),Ac-V-(Orn)-GRT-(1-Nal)-TLC-NH₂,Ac-V-(Orn)-GRT-(1-Nal)-TLC-NH₂ (Cys-Cys dimer),Ac-V-(Orn)-GRT-(1-Nal)-TLCG-NH₂,Ac-V-(Orn)-GRT-(1-Nal)-TLCG-NH₂ (Cys-Cys dimer),Ac-V-(Orn)-GRT-(1-Nal)-TLCGR-NH₂,Ac-V-(Orn)-GRT-(1-Nal)-TLCGR-NH₂ (Cys-Cys dimer),Ac-(Orn)-GRT-(1-Nal)-TLC-(4-Cl-Phe)-NH₂,Ac-(Orn)-GRT-(1-Nal)-TLC--(4-Cl-Phe)-NH₂ (Cys-Cys dimer),Ac-(Orn)-GRT-(1-Nal)-TLC-NH₂,Ac-(Orn)-GRT-(1-Nal)-TLC-NH₂ (Cys-Cys dimer),Ac-KV-(Orn)-GRT-(1-Nal)-TLC-NH₂,Ac-KV-(Orn)-GRT-(1-Nal)-TLC-NH₂ (Cys-Cys dimer),Ac-KVKGRT-(1-Nal)-TLC-NH₂, Ac-KVKGRT-(1-Nal)-TLC-NH₂ (Cys-Cys dimer),Ac-RVKGRT-(1-Nal)-TLC-NH₂, or Ac-RVKGRT-(1-Nal)-TLC-NH₂ (Cys-Cys dimer);

or (ii) the compositions having 40% peptide sequence identity to thecomposition of (i).
 18. A pharmaceutical composition comprising at leastone modified peptide according to claim 1 and a pharmaceuticallyacceptable carrier or excipient.
 19. A method for inhibiting theactivity of at least one enzyme selected from the group consisting ofProtein Kinase B (AKT1), p70S6K, and Abl, AKT1 (PKB alpha), AKT2 (PKBbeta), MAP3K8 (COT), MST4, AURKB (Aurora B), ROCK1, RPS6KB1 (p70S6K),CDC42 BPA (MRCKA), BRAF, RAF1 (cRAF) Y340D Y341D, SGK (SGK1), MAP4K4(HGK), AURKA (Aurora A), AURKC (Aurora C), BRAF V599E, CHEK1 (CHK1),GSG2 (Haspin), CHEK2 (CHK2), FGR, IKBKB (IKK beta), CDK7/cyclin H/MNAT1,and CDC42 BPB (MRCKB), wherein the method comprises contacting the atleast one selected from the enzyme with PKAc, a PKAc fragment, and thevariant PKAc fragment thereof having the amino acid sequence of themodified peptide according to claim 1, thereby inhibiting the activityof the enzyme; or method for inhibiting cell proliferation comprisingcontacting a cell with the pharmaceutical composition according to claim18, wherein proliferation of the cell is inhibited; or a method forpreventing or treating cancer comprising administering to a patienthaving or at risk of having a cancer an effective amount of thepharmaceutical composition according to claim 18, wherein the cancer isprevented or treated; or a method for preventing or treating aneurodegenerative disease or disorder comprising administering to apatient having or at risk of having a neurodegenerative disease ordisorder an effective amount of the pharmaceutical composition accordingto claim 18, wherein the neurodegenerative disease or disorder isprevented or treated; or a method for preventing or treating animmunodeficiency disorder including but not limited to, AIDS, leukemia,lymphoma, viral diseases, hepatitis, multiple myeloma,ataxia-telangiectasia, Chediak-Higashi syndrome, combinedimmunodeficiency disease, complement deficiencies, DiGeorge syndrome,hypogammaglobulinemia, Job syndrome, leukocyte adhesion defects,panhypogammaglobulinemia, Bruton's disease, congenitalagammaglobulinemia, selective deficiency of IgA, and Wiskott-Aldrichsyndrome, wherein the method comprises administering to a patient inneed thereof an effective amount of the pharmaceutical compositionaccording to claim 18.